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Publication Abstracts
MicroRNAs direct rapid deadenylation of mRNA. Wu L, Fan J, Belasco JG Proc. Natl. Acad. Sci. USA 103: 4034-4039 (2006) MicroRNAs (miRNAs) are ubiquitous regulators of eukaryotic gene expression. In addition to repressing translation, miRNAs can down-regulate the concentration of mRNAs that contain elements to which they are imperfectly complementary. Using miR-125b and let-7 as representative miRNAs, we show that in mammalian cells this reduction in message abundance is a consequence of accelerated deadenylation, which leads to rapid mRNA decay. The ability of miRNAs to expedite poly(A) removal does not result from decreased translation; nor does translational repression by miRNAs require a poly(A) tail, a 3' histone stem-loop being an effective substitute. These findings suggest that miRNAs use two distinct posttranscriptional mechanisms to down-regulate gene expression.[back to publication page] Lost in translation: the influence of ribosomes on bacterial mRNA decay. Deana A, Belasco JG Genes Dev. 19: 2526-2533 (2005) The lifetimes of bacterial mRNAs are strongly affected by their association with ribosomes. Events occurring at any stage during translation, including ribosome binding, polypeptide elongation, or translation termination, can influence the susceptibility of mRNA to ribonuclease attack. Ribosomes usually act as protective barriers that impede mRNA cleavage, but in some instances they can instead trigger the decay of the mRNA to which they are bound or send a signal that leads to widespread mRNA destabilization within a cell. The influence of translation on mRNA decay provides a quality-control mechanism for minimizing the use of poorly or improperly translated mRNAs as templates for the production of abnormal proteins that might be toxic to bacteria.[back to publication page] MicroRNA regulation of the mammalian lin-28 gene during neuronal differentiation of embryonal carcinoma cells. Wu L, Belasco JG Mol. Cell. Biol. 25: 9198-9208 (2005) Vertebrate genomes each encode hundreds of microRNAs (miRNAs), yet for few of these miRNAs is there empirical evidence as to which mRNA(s) they regulate. Here we report the identification of human lin-28 mRNA as a regulatory target of human miR-125b and its homolog miR-125a. Studies of miR-125b function in mouse P19 embryonal carcinoma cells induced to develop into neurons suggest a role for this regulatory miRNA in mammalian neuronal differentiation, since its increased concentration in these cells contributes to lin-28 downregulation. Within the lin-28 3' untranslated region (UTR) are two conserved miRNA responsive elements (miREs) that mediate repression by miR-125b and miR-125a. Simultaneous deletion of both miREs renders the lin-28 3' UTR almost completely insensitive to these miRNAs, indicating that these two miREs are the principal elements in the lin-28 3' UTR that respond to miR-125. At the 3' end of each element is an adenosine residue that makes a significant contribution to function irrespective of its complementarity to the 5'-terminal nucleotide of miR-125. By contrast to most earlier reports of gene repression by other miRNAs that are imperfectly complementary to their targets, lin-28 downregulation by miR-125 involves reductions in both translational efficiency and mRNA abundance. The decrease in the mRNA concentration is achieved by a posttranscriptional mechanism that is independent of the inhibitory effect on translation.[back to publication page] Catalytic activation of multimeric RNase E and RNase G by 5'-monophosphorylated RNA. Jiang X, Belasco JG Proc. Natl. Acad. Sci. USA 101: 9211-9216 (2004) RNase E is an endonuclease that plays a central role in RNA processing and degradation in Escherichia coli. Like its E. coli homolog RNase G, RNase E shows a marked preference for cleaving RNAs that bear a monophosphate, rather than a triphosphate or hydroxyl, at the 5' end. To investigate the mechanism by which 5'-terminal phosphorylation can influence distant cleavage events, we have developed fluorogenic RNA substrates that allow the activity of RNase E and RNase G to be quantified much more accurately and easily than before. Kinetic analysis of the cleavage of these substrates by RNase E and RNase G has revealed that 5' monophosphorylation accelerates the reaction not by improving substrate binding, but rather by enhancing the catalytic potency of these ribonucleases. Furthermore, the presence of a 5' monophosphate can increase the specificity of cleavage site selection within an RNA. Although monomeric forms of RNase E and RNase G can cut RNA, the ability of these enzymes to discriminate between RNA substrates on the basis of their 5' phosphorylation state requires the formation of protein multimers. Among the molecular mechanisms that could account for these properties are those in which 5'-end binding by one enzyme subunit induces a protein structural change that accelerates RNA cleavage by another subunit. [back to publication page] The function of RNase G in Escherichia coli is constrained by its amino and carboxyl termini. Deana A, Belasco JG Mol. Microbiol. 51: 1205-1217 (2004) Despite its importance for RNA processing and degradation in Escherichia coli, little is known about the structure of RNase E or its mechanism of action. We have modeled the three-dimensional structure of an essential amino-terminal domain of RNase E on the basis of its sequence homology to the S1 family of RNA-binding domains. Each of the five surface-exposed aromatic residues and most of the 14 basic residues of this RNase E domain were replaced with alanine to determine their importance for RNase E function. All of the surface residues essential for cell growth and feedback regulation of RNase E synthesis mapped to one end of the domain. In vitro assays indicate that these essential residues fall into two functionally distinct groups that form discrete clusters on opposite faces of the S1 domain. One group, comprising Phe-57, Phe-67, and Lys-112, is of general importance for the ribonuclease activity of RNase E, whereas the other group, comprising Lys-37 and Tyr-60, is entirely dispensable for catalytic activity in vitro. The sidechains of two residues previously identified as sites of temperature-sensitive mutations lie buried directly beneath the surface region defined by Phe-57, Phe-67, and Lys-112, which likely enhances RNase E activity by making a crucial contribution to the binding of substrate RNAs. By contrast to the S1 domain, an arginine-rich RNA-binding domain in the carboxyl half of RNase E appears to have a more peripheral role in RNase E function, as it is not required for feedback regulation, cell growth, or ribonuclease activity. [back to publication page] Two distinct regions on the surface of an RNA-binding domain are crucial for RNase E function. Diwa AA, Jiang X, Schapira M, Belasco JG Mol. Microbiol.46(4): 959-69. (2002) Despite its importance for RNA processing and degradation in Escherichia coli, little is known about the structure of RNase E or its mechanism of action. We have modeled the three-dimensional structure of an essential amino-terminal domain of RNase E on the basis of its sequence homology to the S1 family of RNA-binding domains. Each of the five surface-exposed aromatic residues and most of the 14 basic residues of this RNase E domain were replaced with alanine to determine their importance for RNase E function. All of the surface residues essential for cell growth and feedback regulation of RNase E synthesis mapped to one end of the domain. In vitro assays indicate that these essential residues fall into two functionally distinct groups that form discrete clusters on opposite faces of the S1 domain. One group, comprising Phe-57, Phe-67, and Lys-112, is of general importance for the ribonuclease activity of RNase E, whereas the other group, comprising Lys-37 and Tyr-60, is entirely dispensable for catalytic activity in vitro. The sidechains of two residues previously identified as sites of temperature-sensitive mutations lie buried directly beneath the surface region defined by Phe-57, Phe-67, and Lys-112, which likely enhances RNase E activity by making a crucial contribution to the binding of substrate RNAs. By contrast to the S1 domain, an arginine-rich RNA-binding domain in the carboxyl half of RNase E appears to have a more peripheral role in RNase E function, as it is not required for feedback regulation, cell growth, or ribonuclease activity. [back to publication page] Critical features of a conserved RNA stem-loop important for feedback regulation of RNase E synthesis Diwa AA, Belasco JG J. Biol. Chem. 277: 20415-20422 (2002) RNase E is an important regulatory enzyme that governs the principal pathway for mRNA degradation in Escherichia coli. This endonuclease controls its own synthesis via a feedback mechanism in which the longevity of rne (RNase E) mRNA is modulated by a cis-acting sensory element that responds to changes in cellular RNase E activity. Previous research has shown that this element is an RNA stem-loop (hp2) within the 5'-untranslated region of the rne transcript. Here we report studies involving mutational analysis and phylogenetic comparison that have identified the features of rne hp2 important for its function. These comprise an internal loop flanked on one side by a 2-bp stem and a hairpin loop and on the other side by a longer stem whose sequence is inconsequential. A search of bacterial genome sequences suggests that regulation by an hp2-like element may be a unique evolutionary adaptation of the rne transcript that is not shared by other mRNAs. [back to publication page] Comparative analysis of the plant mRNA-destabilizing element, DST, in mammalian and tobacco cells Feldbrugge M, Aritzi P, Sullivan ML, Zamore PD, Belasco JG, Green PJ Plant Mol. Biol. 49: 215-223 (2002) The labile SAUR transcripts from higher plants contain a conserved DST sequence in their 3'-untranslated regions. Two copies of a DST sequence from soybean are sufficient to destabilize reporter transcripts in cultured tobacco cells whereas variants bearing mutations in the conserved ATAGAT or GTA regions are inactive. To investigate the potential for conserved recognition components in mammalian and plant cells, we examined the function of this instability determinant in mouse NIH3T3 fibroblasts and tobacco BY2 cells. In fibroblasts, a tetrameric DST element from soybean accelerated deadenylation and decay of a reporter transcript. However, a version mutated in the ATAGAT region was equally effective in this regard, and a tetrameric DST element from Arabidopsis was inactive. In contrast, the soybean DST element was more active as an mRNA instability element than the mutant version and the Arabidopsis element, when tested as tetramers in tobacco cells. Hence, the plant DST element is not recognized in animal cells with the same sequence requirements as in plant cells. Therefore, its mode of recognition appears to be plant-specific. [back to publication page] Consequences of RNase E scarcity in Escherichia coli Jain C, Deana A, Belasco JG Mol. Microbiol. 43: 1053-1064 (2002) The endoribonuclease RNase E plays an important role in RNA processing and degradation in Escherichia coli. The construction of an E. coli strain in which the cellular concentration of RNase E can be precisely controlled has made it possible to examine and quantify the effect of RNase E scarcity on RNA decay, gene regulation, and cell growth. These studies show that RNase E participates in a step in the degradation of its RNA substrates that is partially or fully rate-determining. Our data further indicate that E. coli growth requires a cellular RNase E concentration at least 10-20% of normal and that the feedback mechanism that limits overproduction of RNase E is also able to increase its synthesis when its concentration drops below normal. The magnitude of the increase in RNA longevity under conditions of RNase E scarcity may be limited by an alternative pathway for RNA degradation. Additional experiments show that RNase E is a stable protein in E. coli. No other E. coli gene product, either when mutated or when cloned on a multicopy plasmid, seems capable of compensating for an inadequate supply of this essential protein. [back to publication page] T7 phage display: a novel genetic selection system for cloning RNA-binding proteins from cDNA libraries. Danner S, Belasco JG Proc.Natl.Acad.Sci.USA 98: 12954-12959. (2001) RNA-binding proteins are central to posttranscriptional gene regulation and play an important role in a number of major human diseases. Cloning such proteins is a crucial but often difficult step in elucidating the biological function of RNA regulatory elements. To make it easier to clone proteins that specifically bind RNA elements of interest, we have developed a rapid and broadly applicable in vitro genetic selection method based on T7 phage display. Using hairpin II of U1 small nuclear RNA (U1hpII) or the 3' stem loop of histone mRNA as bait, we could selectively amplify T7 phage that display either the spliceosomal protein U1A or the histone stem loop-binding protein from a lung cDNA phage library containing more than 10(7) independent clones. The use of U1hpII mutants with various affinities for U1A revealed that this method allows the selection even of proteins that bind their cognate RNA targets with relatively weak affinities (K(d) as high as the micromolar range). Experiments with a mixture of recombinant phage displaying U1A or the closely related protein U2B" demonstrated that addition of a competitor RNA can suppress selection of a protein with a higher affinity for a given RNA target, thereby allowing the preferential amplification of a lower affinity protein. Together, these findings suggest that T7 phage display can be used to rapidly and selectively clone virtually any protein that binds a known RNA regulatory element, including those that bind with low affinity or that must compete for binding with other proteins. [back to publication page] Structural model for the cooperative assembly of HIV-1 Rev multimers on the RRE as deduced from analysis of assembly-defective mutants. Jain C., Belasco J.G. Mol.Cell 7: 603-614 (2001) The functional efficacy of the HIV-1 Rev protein is highly dependent on its ability to assemble onto its HIV-1 RNA target (the RRE) as a multimeric complex. To elucidate the mechanism of multimeric assembly, we have devised two rapid and broadly applicable strategies for examining cooperative interactions between proteins bound to RNA, one based on cooperative translational repression of a two-site reporter and the other on gel-shift analysis with crude E. coli extracts. Using these strategies, we have identified two distinct surfaces of Rev (head and tail) that are critical for different steps in multimeric assembly. Our data indicate that Rev assembles cooperatively on the RRE via a series of symmetrical tail-to-tail and head-to-head protein-protein interactions. The insights into molecular architecture suggested by these findings have enabled us to derive a structural model for Rev and its multimerization on the RRE. [back to publication page] An evolutionarily conserved RNA stem-loop functions as a sensor that directs feedback regulation of RNase E gene expression. Diwa A., Bricker A.L., Jain C., Belasco J.G. Genes Dev. 14: 1249-1260 (2000) RNase E is a key regulatory enzyme that controls the principal pathway for mRNA degradation in Escherichia coli. The cellular concentration of this endonuclease is governed by a feedback mechanism in which RNase E tightly regulates its own synthesis. Autoregulation is mediated in cis by the 361-nucleotide 5' untranslated region (UTR) of rne (RNase E) mRNA. Here we report the determination of the secondary structure of the rne 5' UTR by phylogenetic comparison and chemical alkylation, together with dissection studies to identify the 5' UTR element that mediates autoregulation. Our findings reveal that the structure and function of the rne 5' UTR are evolutionarily well conserved despite extensive sequence divergence. Within the rne 5' UTR are multiple RNA secondary structure elements, two of which function in cis to mediate feedback regulation of rne gene expression. The more potent of these two elements is a stem-loop structure containing an internal loop whose sequence is the most highly conserved of any region of the rne 5' UTR. Our data show that this stem-loop functions as a sensor of cellular RNase E activity that directs autoregulation by modulating the degradation rate of rne mRNA in response to changes in RNase E activity. [back to publication page] Regions of RNase E important for 5'-end-dependent RNA cleavage and autoregulated synthesis. Jiang X., Diwa A., Belasco J.G. J. Bacteriol. 182: 2468-2475 (2000) RNase E is an important regulatory enzyme that plays a key role in RNA processing and degradation in E. coli. Internal cleavage by this endonuclease is accelerated by the presence of a monophosphate at the RNA 5' end. Here we show that the preference of E. coli RNase E for 5' monophosphorylated substrates is an intrinsic property of the catalytically active amino-terminal half of the enzyme and does not require the carboxy-terminal region. This property is shared by the related E. coli ribonuclease CafA (RNase G) and by a cyanobacterial RNase E homolog derived from Synechocystis sp., indicating that the 5'-end dependence of RNase E is a general characteristic of members of this ribonuclease family, including those from evolutionarily distant species. Although it is dispensable for 5'-end-dependent RNA cleavage, the carboxy-terminal half of RNase E significantly enhances the ability of this ribonuclease to autoregulate its synthesis in E. coli. Despite similarities in amino acid sequence and substrate specificity, CafA is unable to replace RNase E in sustaining E. coli cell growth or in regulating RNase E production, even when overproduced six-fold relative to wild-type RNase E levels. [back to publication page] Rapid genetic analysis of RNA-protein interactions by translational repression in E. coli. Jain, C., Belasco, J.G. Methods Enzymol. 318: 309-332(2000) [No abstract available] [back to publication page] Importance of a 5' stem-loop for longevity of papA mRNA in Escherichia coli. Bricker, A.L., Belasco, J.G. J. Bacteriol. 181: 3587-3590 (1999) High-level expression of the major pilus subunit (PapA) of uropathogenic strains of Escherichia coli results in part from the unusually long lifetime of the mRNA that encodes this protein. Here we report that the longevity of papA mRNA derives in large measure from the protection afforded by its 5' untranslated region. This papA RNA segment can prolong the lifetime of an otherwise short-lived mRNA to which it is fused. In vivo alkylation studies indicate that, in its natural milieu, the papA message begins with a stem-loop structure. This stem-loop is important for the stabilizing effect of the papA 5' untranslated region, as evidenced by the significant acceleration in papA mRNA decay that results from its removal. [back to publication page] Target descrimination by RNA-binding proteins: role of the ancillary protein U2A' and a critical leucine residue in differentiating the RNA-binding specificity of spliceosomal proteins U1A and U2B". Rimmele M.E., Belasco J.G. RNA 4: 1386-1396 (1998) The spliceosomal proteins U1A and U2B" each use a homologous RRM domain to bind specifically to their respective snRNA targets, U1hpll and U2hpIV, two stem-loops that are similar yet distinct in sequence. Previous studies have shown that binding of U2B" to U2hpIV is facilitated by the ancillary protein U2A', whereas specific binding of U1A to U1hpll requires no cofactor. Here we report that U2A' enables U2B" to distinguish the loop sequence of U2hpIV from that of U1hpll but plays no role in stem sequence discrimination. Although U2A' can also promote heterospecific binding of U1A to U2hpIV, a much higher concentration of the ancillary protein is required due to the approximately 500-fold greater affinity of U2A' for U2B". Additional experiments have identified a single leucine residue in U1A(Leu-44) that is critical for the intrinsic specificity of this protein for the loop sequence of U1 hpll in preference to that of U2hpIV. Our data suggest that most of the difference in RNA-binding specificity between U1A and U2B" can be accounted for by this leucine residue and by the contribution of the ancillary protein U2A' to the specificity of U2B". [back to publication page] mRNA stabilization by the ompA 5' untranslated region: two protective elements hinder distinct pathways for mRNA degradation. Arnold T.E., Yu J., Belasco J.G. RNA 4: 319-330 (1998) The 5' untranslated region (UTR) of the long-lived Escherichia coli ompA transcript functions as an mRNA stabilizer that can prolong the cytoplasmic lifetimes of a variety of messages to which it is fused. Previous studies have identified two domains of this 5' UTR that together are responsible for its stabilizing effect. One is a 5'-terminal stem-loop. The other is a single-stranded RNA segment (ss2) that contains a ribosome binding site highly complementary to 16S ribosomal RNA. Here we report a detailed investigation of the function of these two stabilizing elements. Our data indicate that mRNA protection by a 5' stem-loop requires no sequence features or thermodynamic stability beyond the minimum necessary for stem-loop formation. Stabilization by ss2 appears to result not from a high frequency of translation initiation, but rather from a high degree of occupancy of this 5' UTR segment by bound ribosomes. Although close spacing of translating ribosomes is not critical for message stabilization by the ompA 5' UTR, mRNA longevity does require the periodic passage of ribosomes through the protein-coding region. Unlike bound ribosomes, which hinder mRNA cleavage by RNase E, the 5' stem-loop appears to impede degradation of ompA mRNA via a distinct pathway that is RNase E-independent. These findings imply that the ompA 5' UTR prolongs mRNA longevity by impeding multiple pathways for mRNA degradation. [back to publication page] RNA binding proteins tamed. Laird-Offringa I.A., Belasco J.G. Nat. Struct. Biol. 5: 665-668 (1998) Novel RNA-binding proteins with customized specificities can be isolated by genetic selection from combinatorial libraries. Such proteins have great potential as agents for targeted manipulation of gene expression. [back to publication page] RNA recognition by the joint action of two nucleolin RNA-binding domains: genetic analysis and structural modeling. Bouvet P., Jain C., Belasco J.G., Amalric F., Erard M. (1997) EMBO J, 16: 5235-5246 (1997) The interaction of nucleolin with a short stem-loop structure (NRE) requires two contiguous RNA-binding domains (RBD 1+2). The structural basis for RNA recognition by these RBDs was studied using a genetic system in Escherichia coli. Within each of the two domains, we identified several mutations that severely impair interaction with the RNA target. Mutations that alter RNA-binding specificity were also isolated, suggesting the identity of specific contacts between RBD 1+2 amino acids and nucleotides within the NRE stem-loop. Our data indicate that both RBDs participate in a joint interaction with the NRE and that each domain uses a different surface to contact the RNA. The constraints provided by these genetic data and previous mutational studies have enabled us to propose a three-dimensional model of nucleolin RBD 1+2 bound to the NRE stem-loop. [back to publication page] A rapid genetic method for the study of RNA binding proteins. Jain, C., Belasco, J.G. In: Richter JD, ed., mRNA Formation and Function Academic Press: 263-284 (1997) [No abstract available] [back to publication page] A structural model for the HIV-1 Rev-RRE complex deduced from altered-specificity rev variants isolated by a rapid genetic strategy. Jain, C., Belasco, J.G. Cell87: 115-125 (1996) A broadly applicable genetic strategy was developed for investigating RNA-protein interactions and applied to the HIV-1 Rev protein. By rapidly screening thousands of Rev-RNA interactions in Escherichia coli, we isolated Rev suppressor mutations that alleviated the deleterious effect of mutations in RRE stem-loop IIB, the high affinity RNA-binding site for Rev. All of these suppressor mutations map to a single arginine-deficient face of a Rev alpha-helix, and some alter the binding specificity of the protein, providing genetic evidence for direct contacts between specific Rev amino acids and RNA nucleotides in the RNA complex of Rev. The spatial constraints suggested by these data have enabled us to model the structure of this complex. [back to publication page] Translation of the adhE transcript to produce ethanol dehydrogenase requires RNase III cleavage in Escherichia coli Aristarkhov A., Mikulskis A., Belasco J.G., Lin E.C.C. J. Bacteriol.178: 4327-4332 (1996) Previous studies have shown that the adhE gene, which encodes a multifunctional protein with ethanol dehydrogenase activity, is under transcriptional regulation. The level of dehydrogenase activity in cells grown fermentatively is about 10-fold higher than that in cells grown aerobically. In these studies, we mapped the promoter to a region well upstream of the protein-coding region of adhE. Unexpectedly, in mutants lacking the endoribonuclease RNase III, no significant ethanol dehydrogenase activity was detected in cells grown anaerobically on rich (Luria-Bertani) medium supplemented with glucose, even though adhE mRNA levels were high. Indeed, like Delta adhE mutants, strains lacking RNase III failed to grow fermentatively on glucose but grew on the more oxidized carbon source glucuronate. Computer-generated secondary structures of the putative 5' untranslated region of adhE mRNA suggest that the ribosome binding site is occluded by intramolecular base pairing. It seems likely that cleavage of this secondary structure by RNase III is necessary for efficient translation initiation.. [back to publication page] In vitro genetic analysis of RNA-binding proteins using phage display libraries. Laird-Offringa I.A., Belasco J.G. Methods Enzymol.267: 149-168 (1996) [No abstract available] [back to publication page] Analysis of RNA-binding proteins by in vitro genetic selection: identification of an amino acid residue important for locking U1A onto its RNA target Laird-Offringa I.A., Belasco J.G. Proc. Natl. Acad. Sci. USA92: 11859-11863 (1995) An in vitro genetic system was developed as a rapid means for studying the specificity determinants of RNA-binding proteins. This system was used to investigate the origin of the RNA-binding specificity of the mammalian spliceosomal protein U1A. The U1A domain responsible for binding to U1 small nuclear RNA was locally mutagenized and displayed as a combinatorial library on filamentous bacteriophage. Affinity selection identified four U1A residues in the mutagenized region that are important for specific binding to U1 hairpin II. One of these residues (Leu-49) disproportionately affects the rates of binding and release and appears to play a critical role in locking the protein onto the RNA. Interestingly, a protein variant that binds more tightly than U1A emerged during the selection, showing that the affinity of U1A for U1 RNA has not been optimized during evolution. [back to publication page] The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation. Zubiaga A.M., Belasco J.G., Greenberg M.E. Mol. Cell. Biol15: 2219-2230 (1995) Labile mRNAs that encode cytokine and immediate-early gene products often contain AU-rich sequences within their 3' untranslated region (UTR). These AU-rich sequences appear to be key determinants of the short half-lives of these mRNAs, although the sequence features of these elements and the mechanism by which they target mRNAs for rapid decay have not been fully defined. We have examined the features of AU-rich elements (AREs) that are crucial for their function as determinants of mRNA instability in mammalian cells by testing the ability of various mutant c-fos AREs and synthetic AREs to direct rapid mRNA deadenylation and decay when inserted within the 3' UTR of the normally stable beta-globin mRNA. Evidence is presented that the pentamer AUUUA, which previously was suggested to be the minimal determinant of instability present in mammalian AREs, cannot direct rapid mRNA deadenylation and decay. Instead, the nonomer UUAUUUAUU is the elemental AU-rich sequence motif that destabilizes mRNA. Removal of one uridine residue from either end of the nonamer (UUAUUUAU or UAUUUAUU) results in a decrease of potency of the element, while removal of a uridine residue from both ends of the nonamer (UAUUUAU) eliminates detectable destabilizing activity. The inclusion of an additional uridine residue at both ends of the nonamer (UUUAUUUAUUU) does not further increase the efficacy of the element. Taken together, these findings suggest that the nonamer UUAUUUAUU is the minimal AU-rich motif that effectively destabilizes mRNA. Additional ARE potency is achieved by combining multiple copies of this nonamer in a single mRNA 3' UTR. Furthermore, analysis of poly(A) shortening rates for ARE-containing mRNAs reveals that the UUAUUUAUU sequence also accelerates mRNA deadenylation and suggests that the UUAUUUAUU motif targets mRNA for rapid deadenylation as an early step in the mRNA decay process. [back to publication page] Autoregulation of RNase E synthesis in Escherichia coli Jain C., Belasco J.G. Nucleic Acids Symp. Ser.33: 85-88 (1995) RNase E plays a central role in controlling mRNA degradation in E. coli. We have investigated the mechanism of RNase E autoregulation. Our data indicate that RNase E autoregulates its synthesis by controlling the decay rate of its own transcript (rne mRNA), which is unusually sensitive to the level of cellular RNase E activity. Feedback regulation is mediated in cis by the rne 5' untranslated region (5' UTR), which can confer this property onto heterologous mRNAs to which it is fused. The marked sensitivity of rne mRNA to regulation by RNase E is also due in part to the susceptibility of nascent rne transcripts to RNase E-mediated degradation. [back to publication page] RNase E autoregulates its synthesis by controlling the degradation rate of its own mRNA in Escherichia coli: unusual sensitivity of the rne transcript to RNase E activity Jain C., Belasco J.G. Genes Dev.9: 84-96 (1995) RNase E is a key regulatory enzyme that appears to control the principal pathway for mRNA degradation in Escherichia coli. Here, we show that RNase E represses its own synthesis by reducing the cellular concentration of the rne (RNase E) gene transcript. Autoregulation is achieved by modulating the longevity of this 3.6-kb mRNA, whose half-life ranges from < 40 sec to 8 min depending on the level of RNase E activity in the cell. Feedback regulation is mediated in cis by the 5'-terminal 0.44-kb segment of rne mRNA, which is sufficient to confer this property onto a heterologous transcript to which it is fused. Like the intact protein, an amino-terminal fragment of RNase E lacking 563 amino acid residues can act in trans to repress rne gene expression. Paradoxically, raising the rne gene copy number 21-fold in E. coli causes an unexpected reduction in the concentration of the full-length rne transcript, yet results in a small increase in RNase E protein production. These surprising phenomena are explained in terms of a model in which the degradation of this long and highly labile mRNA commences before elongation of the nascent transcript has been completed. In such circumstances, gene expression can be unusually sensitive to changes in mRNA stability. [back to publication page] The ompA 5' untranslated region impedes a major pathway for mRNA degradation in Escherichia coli Hansen M.J., Chen L.H., Fejzo M.L., Belasco J.G. Mol. Microbiol.12: 707-716 (1994) The unusual longevity of the Escherichia coli ompA transcript is determined by its 5' untranslated region (UTR), which functions in vivo as an mRNA stabilizer. Here we show that this 5' UTR can prolong the lifetime in E. coli of a variety of heterologous mRNAs to which it is joined, either as a gene fusion or as an operon fusion. Statistical extrapolation suggests that it is quite likely that most E. coli mRNAs could be stabilized in this manner. We conclude that the ompA 5' UTR impedes a major pathway for mRNA degradation in E. coli and that stabilization by fusion to this UTR does not require translational readthrough of the heterologous mRNA segment by ribosomes that initiate translation at the ompA ribosome-binding site. Additional experiments indicate that the E. coli ribonuclease whose action is slowed by the ompA 5' UTR is not RNase III. [back to publication page] Multiple elements in the c-fos protein-coding region facilitate mRNA deadenylation and decay by a mechanism coupled to translation. Schiavi S.C., Wellington C.L., Shyu A.B., Chen C.Y., Greenberg M.E., Belasco J.G. J. Biol. Chem.269: 3441-3448 (1994) The c-fos proto-oncogene transcript is one of the most labile mammalian mRNAs known. Rapid degradation of c-fos mRNA is mediated by both the c-fos protein-coding region and an AU-rich element in the 3'-untranslated region. Here we present evidence that the c-fos coding region contains multiple destabilizing elements that can function independently to facilitate both deadenylation and decay of mRNA. The ability of these coding region destabilizing elements to direct deadenylation and decay requires the assembly of ribosomes at the 5' end of this domain and, most likely, translation of the message. [back to publication page] The destabilizing elements in the coding region of c-fos mRNA are recognized as RNA. Wellington C.L., Greenberg M.E., Belasco J.G. Mol. Cell. Biol.13: 5034-5042. (1993) The protein-coding region of the c-fos proto-oncogene transcript contains elements that direct the rapid deadenylation and decay of this mRNA in mammalian cells. The function of these coding region instability determinants requires movement of ribosomes across mRNAs containing them. Three types of mechanisms could account for this translational requirement. Two of these possibilities, (i) that rapid mRNA decay might be mediated by the nascent polypeptide chain and (ii) that it might result from an unusual codon usage, have experimental precedent. Here, we present evidence that the destabilizing elements in the c-fos coding region are not recognized in either of these two ways. Instead, the ability of the c-fos coding region to function as a potent mRNA destabilizer when translated in the +1 reading frame indicates that the signals for rapid deadenylation and decay reside in the sequence or structure of the RNA comprising this c-fos domain. [back to publication page] Regulation of proto-oncogene mRNA stability. Schiavi S.C., Belasco J.G., Greenberg M.E. Biochim. Biophys. Acta1114: 95-106 (1992) NO ABSTRACT AVAILABLE. [back to publication page] Control of RNase E-mediated RNA degradation by 5'-terminal base pairing in E. coli Bouvet P., Belasco J.G. Nature360:488-491 (1992) Despite the variety of messenger RNA half-lives in bacteria (0.5-30 min in Escherichia coli) and their importance in controlling gene expression, their molecular basis remains obscure. The lifetime of an entire mRNA molecule can be determined by features near its 5' end, but no 5' exoribonuclease has been identified in any prokaryotic organism. A mutation that inactivates E. coli RNase E also increases the average lifetime of bulk E. coli mRNA and of many individual messages, suggesting that cleavage by this endonuclease may be the rate-determining step in the degradation of most mRNAs in E. coli. We have investigated the substrate preference of RNase E in E. coli by using variants of RNA I, a small untranslated RNA whose swift degradation in vivo is initiated by RNase E cleavage at an internal site. We report here that RNase E has an unprecedented substrate specificity for an endoribonuclease, as it preferentially cleaves RNAs that have several unpaired nucleotides at the 5' end. The sensitivity of RNase E to 5'-terminal base pairing may explain how determinants near the 5' end can control rates of mRNA decay in bacteria. [back to publication page] A 5'-terminal stem-loop structure can stabilize mRNA in Escherichia coli Emory S.A., Bouvet P., Belasco J.G. Genes Dev.6: 135-148 (1992) The 5'-untranslated region of the long-lived Escherichia coli ompA transcript functions as an mRNA stabilizer capable of prolonging the lifetime in E.coli of a number of heterologous messages to which it is fused. To elucidate the structural basis of differential mRNA stability in bacteria, the domains of the ompA 5'-untranslated region that allow it to protect mRNA from degradation have been identified by mutational analysis. The presence of a stem-loop no more than 2-4 nucleotides from the extreme 5' terminus of this RNA segment is crucial to its stabilizing influence, whereas the sequence of the stem-loop is relatively unimportant. The potential to form a hairpin very close to the 5' end is a feature common to a number of stable prokaryotic messages. Moreover, the lifetime of a normally labile message (bla mRNA) can be prolonged in E. coli by adding a simple hairpin structure at its 5' terminus. Accelerated degradation of ompA mRNA in the absence of a 5'-terminal stem-loop appears to start downstream of the 5' end. We propose that E. coli messages beginning with a single-stranded RNA segment of significant length are preferentially targeted by a degradative ribonuclease that interacts with the mRNA 5' terminus before cleaving internally at one or more distal sites. [back to publication page] Structure and function of a bacterial mRNA stabilizer: analysis of the 5' untranslated region of ompA mRNA. Chen L.H., Emory S.A., Bricker A.L., Bouvet P., Belasco J.G. J. Bacteriol.173: 4758-4586 (1991) The 5' untranslated region (UTR) of the Escherichia coli ompA transcript functions in vivo as a growth rate-regulated mRNA stabilizer. The secondary structure of this mRNA segment has been determined by a combination of three methods: phylogenetic analysis, in vitro probing with a structure-specific RNase, and methylation by dimethylsulfate in vivo and in vitro. These studies reveal that despite extensive sequence differences, the 5' UTRs of the ompA transcripts of E. coli, Serratia marcescens, and Enterobacter aerogenes can fold in a remarkably similar fashion. Furthermore, the Serratia and Enterobacter ompA 5' UTRs function as effective mRNA stabilizers in E. coli. Stabilization of mRNA by the Serratia ompA 5' UTR is growth rate dependent. These findings indicate that the features of the ompA 5' UTR responsible for its ability to stabilize mRNA in a growth rate-regulated manner are to be found among the structural similarities shared by these diverse evolutionary variants. [back to publication page] Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid mRNA decay. Shyu A.B., Belasco J.G., Greenberg M.E. Genes Dev. 5: 221-231 (1991) The 5' untranslated region (UTR) of the Escherichia coli ompA transcript functions in vivo as a growth rate-regulated mRNA stabilizer. The secondary structure of this mRNA segment has been determined by a combination of three methods: phylogenetic analysis, in vitro probing with a structure-specific RNase, and methylation by dimethylsulfate in vivo and in vitro. These studies reveal that despite extensive sequence differences, the 5' UTRs of the ompA transcripts of E. coli, Serratia marcescens, and Enterobacter aerogenes can fold in a remarkably similar fashion. Furthermore, the Serratia and Enterobacter ompA 5' UTRs function as effective mRNA stabilizers in E. coli. Stabilization of mRNA by the Serratia ompA 5' UTR is growth rate dependent. These findings indicate that the features of the ompA 5' UTR responsible for its ability to stabilize mRNA in a growth rate-regulated manner are to be found among the structural similarities shared by these diverse evolutionary variants. [back to publication page] Degradation of pufLMX mRNA in Rhodobacter capsulatus is initiated by nonrandom endonucleolytic cleavage. Chen C.Y., Belasco J.G. J. Bacteriol.172: 4578-4586 (1990) Differential expression of genes within the puf photosynthesis operon of Rhodobacter capsulatus is achieved primarily through marked segmental differences in stability within the polycistronic puf operon transcripts.The comparatively stable pufBA segment of these transcripts outlives the labile pufLMX segment and accumulates as an abundant puf mRNA degradation intermediate. Here we present further evidence that degradation of pufBALMX mRNA is initiated by endonucleolytic cleavage within the short-lived pufLMX mRNA segment. By deletion analysis, a region sufficient to mediate rapid degradation of this labile RNA segment has been defined. The 3' boundary of this region maps to within a stretch of 30 nucleotides corresponding to pufL codons 49 through 59. Evidence that initial cleavage of the pufLMX RNA segment occurs predominantly upstream of pufM codon 99 has been obtained by using a novel method, hairpin insertion analysis. Additional data indicate that the efficacy of RNA stem-loop structures as 3'-exonuclease barriers is reduced when they are located in translated regions of messages. [back to publication page] The ompA 5'untranslated RNA segment functions in Escheria coli as a growth-rate-regulated mRNA stabilizer whose activity is unrelated to translational efficiency. Emory, SA, Belasco, JG J. Bacteriol.172: 4472-4481 (1990) The 5' untranslated region (UTR) of the long-lived Escherichia coli ompA message can function in vivo as an mRNA stabilizer. Substitution of this ompA mRNA segment for the corresponding segment of the labile bla gene transcripts prolongs their lifetime by a factor of 6. We show here that the function of this ompA mRNA stabilizer requires the presence of a 115-nucleotide ompA RNA segment that lies upstream of the ribosome-binding site. Although deletion of this segment reduced the half-life of the ompA transcript by a factor of 5, its absence had almost no effect on the translational efficiency of ompA mRNA. Like the ompA transcript, but unlike bla mRNA, hybrid ompA-bla messages containing the complete ompA 5' UTR were significantly less stable under conditions of slow bacterial growth. We conclude that the stabilizing activity of the ompA 5' UTR is growth rate regulated and that the mechanism of mRNA stabilization by this RNA segment is not related to the spacing between translating ribosomes. [back to publication page] Deadenylylation: a mechanism controlling c-fos mRNA decay. Greenberg ME, Shyu AB, Belasco JG Enzyme44:181-92 (1990) The c-fos proto-oncogene mRNA is extremely labile and is rapidly degraded within minutes after being transported to the cytoplasm of growth factor-stimulated fibroblasts. Analysis of the structural determinants controlling c-fos message decay reveals that this message contains at least two functionally independent elements that are responsible for its short half-life. One of these determinants is an AU-rich sequence present in the 3' untranslated region of the c-fos message, whereas the other determinant, which is structurally unrelated to the AU-rich element, is located within the c-fos protein-coding sequence. Both the c-fos AU-rich element and the coding region instability determinant appear to function by facilitating rapid removal of the c-fos poly(A) tail as a first step in the mRNA degradation process. [back to publication page] The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways. Shyu AB, Greenberg ME, Belasco JG Genes Dev3:60-72 (1989) Rapid degradation of c-fos proto-oncogene mRNA is crucial for transient c-fos gene expression. Experiments were performed to investigate the cellular mechanisms responsible for the extremely short half-life of human c-fos mRNA in growth-factor-stimulated fibroblasts. These experiments demonstrate the existence of two distinct cellular pathways for rapid c-fos mRNA degradation. Each of these pathways recognizes a different, functionally independent instability determinant within the c-fos transcript. One instability determinant, which is located within the c-fos 3'-untranslated region, is a 75-nucleotide AU-rich segment. Insertion of this element into beta-globin mRNA markedly reduces the half-life of that normally long-lived message. Nevertheless, specific deletion of the AU-rich element from c-fos mRNA has little effect on the transcript's cytoplasmic half-life due to the presence of the other c-fos instability determinant, which is located in the protein-coding segment of the c-fos message. Examination of mRNA decay in cells treated with transcription inhibitors indicates that one c-fos mRNA degradation pathway is dependent on RNA synthesis, whereas the other is not. [back to publication page] Mechanism of puf mRNA degradation: the role of an intercistronic stem-loop structure. Belasco JG, Chen CY Genes Dev72:109-117 (1988) The puf photosynthesis operon of Rhodobacter capsulatus encodes two major classes of mRNA: operon-length pufBALMX transcripts and short pufBA messages. The pufBA messages, which end in a large intercistronic stem-loop structure, are long-lived processing products of the puf operon transcripts. Decay of the labile pufLMX segment of the operon-length transcripts begins with non-random endonucleolytic cleavage well downstream of the intercistronic hairpin structure. This hairpin, which is necessary but insufficient for the stability of the RNA segment upstream of it, appears to function as an mRNA decay terminator that protects the upstream pufBA segment from 3' exonucleolytic propagation of the initial degradative event. The comparative stability of the pufBA mRNA segment depends not only on the presence of this stem-loop structure, but also on the relative resistance of the pufBA segment to endonuclease attack. [back to publication page] Mechanisms of mRNA decay in bacteria: a perspective. Belasco JG, Higgins CF Gene72:15-23 (1988) Messenger RNA decay plays an important role in prokaryotic gene expression. The disparate stabilities of bacterial messages in vivo are a consequence of their differential susceptibility to degradation by cellular endoribonucleases and 3' -exoribonucleases, which in turn results from differences in mRNA sequence and structure. RNase II and polynucleotide phosphorylase, the major bacterial exonucleases involved in mRNA turnover, rapidly degrade single-stranded RNA from the 3' end, but are impeded by 3' stem-loop structures. At present, the identify and substrate specificity of the endonucleases that control mRNA decay rates are relatively poorly defined. Ribosomes and antisense RNA also can influence the stability of transcripts with which they associate. Differences in mRNA stability can contribute to differential expression of genes within polycistronic operons and to modulation of gene expression in response to changes in bacterial growth conditions. [back to publication page] An intercistronic stem-loop structure functions as an mRNA decay terminator necessary but insufficient for puf mRNA stability. Chen CY, Beatty JT, Cohen SN, Belasco JG Cell52: 609-619 (1988) Segmental differences in stability within the polycistronic transcripts of the puf operon contribute to differential expression of photosynthesis genes in R. capsulatus. The comparatively stable 5' segment of these transcripts ends in a large intercistronic stem-loop structure. We show here that deletion of this RNA hairpin destabilizes the 5' puf mRNA segment but that its insertion at the 3' end of the puf operon transcripts fails to stabilize the labile 3' puf mRNA segment. Evidence is presented that decay of the 3' segment begins with endonucleolytic cleavage in which the intercistronic stem-loop structure does not participate. We conclude that this RNA hairpin is necessary but insufficient for the stability of mRNA upstream of it, and that it functions in message degradation solely as an mRNA decay terminator that protects upstream mRNA segments from degradation by 3' exoribonucleases. [back to publication page] Effect of premature termination of translation on mRNA stability depends on the site of ribosome release. Nilsson G, Belasco JG, Cohen SN, von Gabain A Proc Natl Acad Sci U S A84:4890-4894 (1987) Translational stop codons were introduced at various locations in the protein-coding regions of the monocistronic bla and ompA gene transcripts of Escherichia coli, and the decay characteristics of the upstream and downstream mRNA segments were analyzed. Premature termination of translation at codon position 26 reduced the stability of both the translated and ribosome-free segments of bla mRNA, whereas release of ribosomes just 30 codons further downstream resulted in normal stability for both segments. Normal stability of an untranslated bla gene mRNA segment required its linkage to a ribosome-bound segment of bla gene mRNA. These findings indicate that depriving an mRNA segment of ribosomes does not necessarily render it more susceptible to degradation. However, premature termination of translation at a location that allows ribosomes to traverse only a short segment of bla mRNA can lead to destabilization of the entire transcript. [back to publication page] The stability of E. coli gene transcripts is dependent on determinants localized to specific mRNA segments. Belasco JG, Nilsson G, von Gabain A, Cohen S Cell46:245-251 (1986) To map the structural features responsible for the 5-fold difference in stability of the E. coli ompA and bla gene transcripts, we have constructed gene fusions that encode chimeric ompA/bla transcripts and a deletion that eliminates a large internal segment of bla mRNA. Shortening of bla transcripts by internal deletion or replacement of the 3' end with the corresponding segment of the ompA transcript had little effect on bla mRNA stability. However, fusion of a 5'-terminal 147 nucleotide segment of the ompA message 5' to full-length or truncated bla transcripts increased the half-life of the bla segments 3- to 5-fold. These and other findings indicate that E. coli transcripts contain discrete structural determinants of stability and instability that can influence the decay rate of linked mRNA segments derived from other genes. [back to publication page] Energetics of proline racemase: rates, fractionation factors, and buffer catalysis in the oversaturated region. Nature of the interconversion of the two forms of free enzyme. Belasco JG, Bruice TW, Fisher LM, Albery WJ, Knowles JR Biochemistry25:2564-71 (1986) To probe the nature of the interconversion of the two unliganded forms of proline racemase, a number of experiments have been performed under oversaturating conditions where the rate of the enzymic reaction is mainly limited by the rate of this interconversion. Competitive deuterium washout experiments, where an equimolar mixture of D- and L-proline (in which some or all of one enantiomer is specifically deuterated at the 2-position) is allowed to reach chemical and isotopic equilibrium mediated by the enzyme, have been followed in four ways. The size and the rate of achievement of the maximum perturbation in the optical rotation have been measured, the deuterium content of the substrate at this maximum has been determined, and the final approach to equilibrium after the perturbation maximum has been followed. Further, the enzyme-catalyzed rate of tritium loss from [2-3H]proline has been established. Finally, it has been shown that the enzyme interconversion reaction is catalyzed by several buffers (such as ammonium, hydrazinium, and hydrogen sulfide). These data are discussed in terms of Marcus' theory, which allows a rather detailed picture of the mechanism of free enzyme interconversion to be drawn. This process nicely parallels the mechanism of the enzyme-catalyzed interconversion of the proline enantiomers, and it is evident that substrate racemization (with the concomitant switch of the enzyme-bound protons) is mirrored by the water-mediated switch of the enzyme-bound protons that effects the interconversion of the free enzyme forms. The results favor a stepwise reaction for the interconversion of the free enzyme forms in which a proton is abstracted from a bound water molecule to give a reaction intermediate having a hydroxide ion bound to the diprotonated form of the enzyme. [back to publication page] Energetics of proline racemase: fractionation factors for the essential catalytic groups in the enzyme-substrate complexes. Belasco JG, Bruice TW, Albery WJ, Knowles JR Biochemistry25:2558-2564 (1986) The fractionation factors of protons bound to the essential catalytic groups in proline racemase have been determined by comparison of the time courses of two competitive deuterium washout experiments. The rate of achievement of the maximum perturbation in the optical rotation has been measured in the oversaturated region (that is, at high substrate concentrations) under two conditions: in the first, we start with an equimolar mixture of deuterated substrate S' and of unlabeled product P; in the second, we again start with equal concentrations of substrate and product, but the concentration of the deuterated material S' is less than 20% that of S. The different concentrations of deuterated substrate produce different levels of deuteration of the enzyme's catalytic groups, the kinetic consequence of which allow the fractionation factors of these enzymic groups to be determined. The observed values for the fractionation factors of the enzyme's groups of 0.55 +/- 0.1 are only consistent with these groups' being thiols. This conclusion is supported by results of measurements of the solvent isotope effect determined in the unsaturated regime. These findings confirm the earlier suggestion of Abeles and his group that two cysteine residues mediate the catalysis of proline racemization by this enzyme. [back to publication page] Energetics of proline racemase: double fractionation experiment, a test for concertedness and for transition-state dominance. Belasco JG, Albery WJ, Knowles JR Biochemistry25:2552-2558 (1986) To test whether a reaction involving the making and/or breaking of two bonds at two sites is concerted (and proceeds through a single transition state) or is stepwise (and involves a reaction intermediate in which only one bond has been made or broken), we have measured the isotopic fractionation at one site as a function of isotopic substitution at the other site. In the case of proline racemase, the discrimination against solvent deuterium in the product when the reaction is run in mixed H2O-D2O is measured for the reaction both of [2-1H]proline and of [2-2H]proline. The isotopic fractionation at the solvent site may in principle be smaller, the same, or larger, when the 2H-labeled substrate is used rather than the 1H substrate, and--depending upon the nature of the catalyzing groups--this information indicates whether the reaction is stepwise, or concerted, or whether an isotopically insensitive transition state is partially rate determining. Experimentally, we have found that the discrimination against solvent deuterium in the product L-proline is the same, whether D-[2-1H]proline or D-[2-2H]proline is the substrate. This result requires that the substrate and product "on-off" steps are faster than the racemization step and that the racemization reaction proceeds either in a concerted manner or in a stepwise fashion involving enzyme catalytic groups (e.g., thiols) having ground-state fractionation factors around 0.5. [back to publication page] Energetics of proline racemase: transition-state fractionation factors for the two protons involved in the catalytic steps. Fisher LM, Belasco JG, Bruice TW, Albery WJ, Knowles JR Biochemistry25:2543-2551 (1986) The isotope effects for the interconversion of L-proline and D-proline, catalyzed by proline racemase, have been determined in the saturated region with both [2-2H]proline and [2-3H]proline. The deuterium fractionation factors for each of the protons in flight have been obtained from two kinds of experiment: by measuring the rate of racemization of one [2-2H]proline enantiomer as it racemizes into an equilibrated pool of unlabeled proline and by measuring the deuterium content of a proline sample at the optical rotation maximum that occurs when an equimolar mixture of one deuterium-labeled enantiomer and the other unlabeled enantiomer runs to equilibrium. The tritium fractionation factors for each of the protons in flight have been determined from measurements of the rate of loss of tritium to the solvent as one [2-3H]proline enantiomer runs to equilibrium. Good agreement is found among the fractionation factors determined by each method. The deuterium fractionation factors for the two protons are not identical: that for the proton derived from L-proline is 0.375 and that for the proton derived from D-proline is 0.44. This difference has been confirmed by a double-competition experiment in which the optical rotation of a mixture of DL-[2-2H]proline and unlabeled DL-proline is followed with time. The rotation (initially zero) passes through a maximum, from which the ratio of the two fractionation factors (0.86) is obtained. These data, coupled with the equilibrium fractionation factor for the 2-position of proline (which has been determined to be 1.17), provide the transition-state factors for each of the in-flight protons, and delineate the nature of the transition state(s) for the enzyme-catalyzed racemization. [back to publication page] Differential expression of photosynthesis genes in R. capsulata results from segmental differences in stability within the polycistronic rxcA transcript. Belasco JG, Beatty JT, Adams CW, von Gabain A, Cohen SN Cell40:171-181 (1985) We report that the light-harvesting and reaction center genes in the rxcA locus of R. capsulata are contained within a single operon and that their differential expression results predominantly from marked segmental differences in stability within the polycistronic rxcA transcript. The 3' portion of this transcript is rapidly degraded to give rise to either of two slowly decaying mRNA remnants, both of which encode only the light-harvesting polypeptides. The greater stability of these remnants accounts for nearly all of the difference between the concentrations of the light-harvesting and reaction center proteins. The unstable 3' portion of the transcript is delimited by two alternative stem-and-loop structures, which apparently act as barriers to 3' exoribonucleases and thereby protect the upstream RNA segment. When a DNA fragment containing the rxcA locus was fused to a plasmid promoter and transcribed in E. coli, the long precursor transcript was processed to two short messages of greater stability, as in R. capsulata. [back to publication page] Growth-rate dependent regulation of mRNA stability in Escherichia coli. Nilsson G, Belasco JG, Cohen SN, von Gabain A Nature312:75-77 (1984) The rate of production of bacterial gene products is known to vary with the rate of cell growth, the concentrations of many cellular proteins are altered during times of decreased growth rate. In addition, proteins whose in vivo levels show no significant alterations with changes in cell doubling time must be synthesized at rates that vary in direct proportion to the growth rate of the cell. In certain instances, growth-rate dependent gene regulation has been shown to occur at the transcriptional or translational level. Another potentially important element in the regulation of gene expression is the stability of messenger RNA. We report here the effect of bacterial growth rate on the half lives of four different monocistronic Escherichia coli mRNA species. The stabilities of two of these species, the transcripts of the ompA and cat genes, exhibited a marked dependence on cell growth rate, whereas the half lives of the transcripts of the lpp and bla genes are constant over a broad range of cell doubling times. Our results indicate that E. coli can alter the rate of synthesis of certain proteins by modulating mRNA stability in response to changes in the rate of cell growth. [back to publication page] Decay of mRNA in Escherichia coli: investigation of the fate of specific segments of transcripts. von Gabain A, Belasco JG, Schottel JL, Chang AC, Cohen SN Proc Natl Acad Sci U S A80:653-657 (1983) An assay was developed to investigate the fate of specific segments of beta-lactamase (bla) and ompA gene transcripts in Escherichia coli. DNA probes cloned in bacteriophage M13 were treated with an endonuclease capable of cleaving single-stranded DNA, the fragments produced were annealed with total cellular RNA, and the resulting RNA . DNA hybrids were subjected to S1 nuclease treatment and gel fractionation. By using this assay, direct evidence was obtained for 3'-to-5' directionality in the decay of the long-lived mRNA encoded by the ompA gene, and no preferential stability was observed for translated versus untranslated mRNA segments. In the case of bla mRNA, initial cleavage of the full-length transcript was rate limiting, and no decay intermediates were detected. No difference in degradation rate was seen for bla transcripts having variant 3' or 5' termini. [back to publication page] Polarization of substrate carbonyl groups by yeast aldolase: investigation by Fourier transform infrared spectroscopy. Belasco JG, Knowles JR Biochemistry22:122-129 (1983) The infrared spectrum of the complex of D-fructose 1,6-bisphosphate bound to yeast aldolase displays three spectral features between 1700 and 1800 cm-1. One of these (at 1730 cm-1) corresponds to the carbonyl group of enzyme-bound D-fructose 1,6-bisphosphate and/or dihydroxyacetone phosphate. The frequency of this band, which is unaffected by the removal of the intrinsic zinc ion from the enzyme, demonstrates that this carbonyl group is not significantly polarized when the substrate binds to the enzyme. In contrast, the spectral band assigned to the carbonyl group of enzyme-bound D-glyceraldehyde 3-phosphate (at 1706 cm-1) appears at a frequency 24 cm-1 lower than when this substrate is in aqueous solution. This shift indicates considerable polarization of the carbonyl group when D-glyceraldehyde 3-phosphate is bound at the active site. The third spectral feature (at 1748 cm-1), which is observed only in the presence of potassium ion, probably corresponds to an enzymic carboxyl group in a nonpolar environment. [back to publication page] beta-Lactamase proceeds via an acyl-enzyme intermediate. Interaction of the Escherichia coli RTEM enzyme with cefoxitin. Fisher J, Belasco JG, Khosla S, Knowles JR Biochemistry19:2895-2801 (1980) The use of cefoxitin, a poor substrate of the RTEM beta-lactamase, has allowed the kinetic and spectroscopic characterization of a covalent acyl-enzyme intermediate in the enzyme-catalyzed reaction. The rate of reappearance of catalytic activity in an enzyme sample diluted from an incubation with cefoxitin is nearly identical with the observed Kcat. Burst kinetics are observed with this substrate, consistent with the rate-limiting deacylation of the cefoxitinoyl-enzyme. That the reaction intermediate involves a covalent link between enzyme and substrate was shown by gel filtration after rapid denaturation of an enzyme-[14C]cefoxitin reaction at the steady state. Fourier transform infrared measurements indicate that the intermediate is an acyl-enzyme involving a hydroxyl group of the beta-lactamase. The evident relationship between the acylation-deacylation sequence of the beta-lactamases and the acylation reaction suffered by the D-Ala-D-Ala-carboxypeptidases is discussed. [back to publication page] Beta-lactamase inactivation by mechanism-based reagents. Fisher J, Belasco JG, Charnas RL, Khosla S, Knowles JR Philos Trans R Soc Lond B Biol Sci289:309-319 (1980) The mechanistic pathway followed by the E. coli RTEM beta-lactamase has been studied with a view to clarifying the mode of action of a number of recently discovered inactivators of the enzyme. There is clear evidence that the beta-lactamase-catalysed hydrolysis of the 7-alpha-methoxycephem, cefoxitin, proceeds via an acyl-enzyme intermediate. An analysis of the inactivation reactions of all the known beta-lactam derivatives that result in irreversible loss of enzyme activity permits the identification of three structural features required for a beta-lactamase inactivator. The application of these principles suggests a new group of mechanism-based inactivators of the enzyme: the sulphones of N-acyl derivatives of 6-beta-aminopenicillanic acid that are themselves poor substrates for the enzyme. These sulphones are powerful inactivators of the beta-lactamase. [back to publication page] Direct observation of substrate distortion by triosephosphate isomerase using Fourier transform infrared spectroscopy. Belasco JG, Knowles JR Biochemistry19:472-477 (1980) The infrared spectrum of dihydroxyacetone phosphate bound to triosephosphate isomerase has been measured. There are two carbonyl bands corresponding to the bound substrate, with an intensity ratio of about 3:1. Relative to the carbonyl absorption of dihydroxyacetone phosphate in free solution, the major band is shifted by 19 cm-1 to 1713 cm-1, providing direct evidence of enzyme-induced distortion of the substrate. This strain is probably attributable to an enzymic electrophile that polarizes the carbonyl group of the substrate and thereby promotes catalysis. [back to publication page] Critical ionization states in the reaction catalyzed by triosephosphate isomerase. Belasco JG, Herlihy JM, Knowles JR Biochemistry17:2971-2978 (1978) To allow the detailed interpretation of the pH dependences of the steady-state parameters for the reaction catalyzed by triosephosphate isomerase, three kinds of experiments have been performed. First, the value of kcat/Km for enzyme-catalyzed isomerization of the phosphonate analogue of D-glyceraldehyde 3-phosphate (2-hydroxy-4-phosphonobutyraldehyde) has been shown to titrate with an apparent pKa of 7.5, which is close to the phosphonate's second ionization constant. Secondly, the sulfate ester analogue of dihydroxyacetone phosphate (dihydroxyacetone sulfate), which exists only as a monoanion over the pH range of interest, has been shown not to bind detectably to the enzyme. Thirdly, an isotopic discrimination experiment at pH 5.2 has been compared with a similar investigation at pH 7.6. The results together demonstrate that both enzyme and substrate ionizations control the reaction rate in the pH range 5 to 8. [back to publication page]
Last Updated: 09-Mar-2006, Daphne Gilles