The ssrA gene encoded tmRNA acts as both a tRNA carrying an Ala to enter the A site of stalled ribosomes and as an mRNA allowing trans-translation to continue until ribosomes reach the stop codon of the tmRNA tag to help release the stalled ribosome, label the truncated peptide for degradation, and also facilitate degradation of the ribosome-stalling mRNAs. Functions of tmRNA rely on its binding to an essential protein factor SmpB that is encoded by the smpB gene. The mycelial bacteria streptomycetes have a well-defined growth and developmental cycle culminating at sporulation and provide a good model to study tmRNA function in bacteria growth and development. During different developmental stages, expression of some critical molecules are increased or decreased to control the developing procedures including a bldA-encoded tRNA that decodes the rare codon UUA. Translation elongation of genes containing UUA rare codons may be stalled and elicits tmRNA tagging, suggesting that tmRNA the tagging system may be important for Streptomyces growth and development. We use the most well studied strain, S. coelicolor whose genome sequence was the first sequenced, as our model organism. Here I report my ssrA knockout study with two different strategies. Using a temperature sensitive replicon, I found that the ssrA gene could be disrupted only in cells with an extra ssrA gene but not in wild type cells or cells with an extra-copy of tmRNA variant--tmRNADD that encodes a degradation-resistant tag. These results imply that ssrA is an essential gene and that degradation of truncated proteins is also an essential function for S. coelicolor. On the contrary, with the second method that does not need high temperature screening steps I was able to disrupt both the ssrA and smpB genes separately and at the same time, suggesting that the tmRNA tagging system may be required for cell survival under high temperature. Further characterization of mutant cells revealed that the tmRNA tagging system is important for cell growth and development at both high temperature and optimal growth conditions as well as under stress conditions that affect the translation elongation process. The second part of my thesis documents analyses of the expression, regulation and stability of S. coelicolor tmRNA. My results suggested that the well known metabolic stability of bacterial tmRNA might be due to its tight binding to the ribosome. Finally, I report my investigation of the tagging activity and the importance of some structural elements of S. coelicolor tmRNA. Particularly, I demonstrated that pseudoknot 4 is important for tmRNA tagging activity and mutations to some structural elements lead to a decrease of not only the mutant tmRNA but also the wild type tmRNA when expressed together in vivo.

The ssrA gene encoded tmRNA acts as both a tRNA carrying an Ala to enter the A site of stalled ribosomes and as an mRNA allowing trans-translation to continue until ribosomes reach the stop codon of the tmRNA tag to help release the stalled ribosome, label the truncated peptide for degradation, and also facilitate degradation of the ribosome-stalling mRNAs. Functions of tmRNA rely on its binding to an essential protein factor SmpB that is encoded by the smpB gene. The mycelial bacteria streptomycetes have a well-defined growth and developmental cycle culminating at sporulation and provide a good model to study tmRNA function in bacteria growth and development. During different developmental stages, expression of some critical molecules are increased or decreased to control the developing procedures including a bldA-encoded tRNA that decodes the rare codon UUA. Translation elongation of genes containing UUA rare codons may be stalled and elicits tmRNA tagging, suggesting that tmRNA the tagging system may be important for Streptomyces growth and development. We use the most well studied strain, S. coelicolor whose genome sequence was the first sequenced, as our model organism. Here I report my ssrA knockout study with two different strategies. Using a temperature sensitive replicon, I found that the ssrA gene could be disrupted only in cells with an extra ssrA gene but not in wild type cells or cells with an extra-copy of tmRNA variant--tmRNADD that encodes a degradation-resistant tag. These results imply that ssrA is an essential gene and that degradation of truncated proteins is also an essential function for S. coelicolor. On the contrary, with the second method that does not need high temperature screening steps I was able to disrupt both the ssrA and smpB genes separately and at the same time, suggesting that the tmRNA tagging system may be required for cell survival under high temperature. Further characterization of mutant cells revealed that the tmRNA tagging system is important for cell growth and development at both high temperature and optimal growth conditions as well as under stress conditions that affect the translation elongation process. The second part of my thesis documents analyses of the expression, regulation and stability of S. coelicolor tmRNA. My results suggested that the well known metabolic stability of bacterial tmRNA might be due to its tight binding to the ribosome. Finally, I report my investigation of the tagging activity and the importance of some structural elements of S. coelicolor tmRNA. Particularly, I demonstrated that pseudoknot 4 is important for tmRNA tagging activity and mutations to some structural elements lead to a decrease of not only the mutant tmRNA but also the wild type tmRNA when expressed together in vivo.

Ribosomes translate the genetic information encoded by mRNA into proteins. Defective mRNAs can cause stalling of translating ribosomes. The molecule tmRNA (transfer-messenger RNA) rescues stalled ribosomes in eubacteria. Together with its protein partner SmpB, tmRNA mimics a tRNA by entering the ribosomal A site and linking an alanine residue to the growing polypeptide chain. The ribosome then abandons the defective mRNA template and resumes translation on tmRNA, adding ten more amino acids to the nascent polypeptide. As a result of tmRNA action, stalled ribosomes are released and recycled, the defective mRNA is destroyed, and the aborted protein product is tagged for destruction by proteases. It is unknown how the ribosome correctly chooses the position on tmRNA to resume translation. Previous studies implicate the sequence UAGUC found immediately upstream of the first codon in the tmRNA open reading frame. These nucleotides are highly conserved in natural tmRNA sequences. Mutations in this area cause loss of tmRNA function and improper frame choice. Using a genetic selection that ties the life of E. coli cells to the function of tmRNA, we have identified several SmpB mutants that rescue an inactive tmRNA in which this upstream sequence was altered. This links SmpB to the function of these key tmRNA nucleotides. We show that our SmpB mutants affect frame choice using an in vivo assay for tagging in the various frames. We conclude that SmpB plays a role in setting the reading frame on tmRNA.

Ribosomes stall on defective messenger RNA transcripts in eubacteria. Without a mechanism to release stalled ribosomes, these cells would die. Transfer-messenger RNA (tmRNA) and small protein B (SmpB) reactivate stalled ribosomes in a process known as trans-translation. Together, tmRNA and SmpB mimic alanyl-tRNA, entering the A site of stalled ribosomes and accepting transfer of the stalled polypeptide. A portion of tmRNA is then positioned as a template for the ribosome to resume translating. The tmRNA open reading frame encodes a proteolysis tag to mark the aberrant polypeptide for degradation and a stop codon to release the ribosome. How are tmRNA and SmpB allowed into stalled ribosomes? In normal translation, decoding mechanisms carefully monitor the anticodon of tRNAs entering the A site and select only those that are complementary to the mRNA codon. How do tmRNA and SmpB get around the decoding machinery? It appears that interactions between the SmpB C-terminal tail and the decoding center are responsible. Using an in vivo tagging assay and an in vitro peptidyl-transfer assay, we monitored the effect of mutations in the SmpB tail on trans-translation. We found that mutations in SmpB that prevent helix formation are unable to support peptidyl transfer. We also found that while mutation of key nucleotides in the ribosomal decoding center severely inhibit peptidyl transfer to normal tRNAs, these mutations do not inhibit peptidyl transfer to tmRNA. We conclude that the SmpB tail stimulates peptidyl transfer by forming a helix that interacts with the ribosome to signal decoding in a novel manner. How is the tmRNA open reading frame positioned for the ribosome to resume translating? Mutation of the tmRNA nucleotide A86 alters reading frame selection. Using a genetic selection, we identified SmpB mutants that restore normal frame selection to A86C tmRNA without altering frame selection on wild-type tmRNA. Through rational mutation of the SmpB tail we identified an SmpB mutant that supports peptidyl transfer but prevents translation of the tmRNA open reading frame. We conclude that SmpB plays a functional role in selecting the tmRNA open reading frame.

Ribosomes translate the genetic information contained in mRNAs into protein by linking together amino acids with the help of aminoacyl-tRNAs. In bacteria, protein synthesis stalls when the ribosome reaches the 3'-end of truncated mRNA transcripts lacking a stop codon. Trans-translation is a conserved bacterial quality control process that rescues stalled ribosomes. Transfer-messenger RNA (tmRNA) and its protein partner SmpB mimic a tRNA by entering the A site of the ribosome and accepting the growing peptide chain. The ribosome releases the truncated mRNA and resumes translation on the tmRNA template. The open reading frame found on tmRNA encodes a peptide tag that marks the defective nascent peptide for proteolysis. A stop codon at the end of the open reading frame allows the ribosome to be recycled and engage in future rounds of translation.The entry of tmRNA into stalled ribosomes presents a challenge to our understanding of ribosome function because during the canonical decoding process, the ribosome specifically recognizes the codon-anticodon duplex formed between tRNA and mRNA in the A site. Recognition of proper base-pairing leads to conformational changes that accelerate GTP hydrolysis by EF-Tu and rapid accommodation of the tRNA into the ribosome for peptidyl transfer. The puzzle is that tmRNA enters stalled ribosomes and reacts with the nascent peptide in the absence of a codon-anticodon interaction. Instead, SmpB binding in the decoding center begins the rescue process, but it has been unclear how SmpB licenses tmRNA entry into stalled ribosomes. We analyzed a series of SmpB and ribosomal RNA mutants using pre-steady-state kinetic assays for EF-Tu activation and peptidyl transfer. Although the conserved 16S nucleotides A1492 and A1493 play an essential role in canonical decoding, they play little or no role in EF-Tu activation or peptidyl transfer to tmRNA. In contrast, a third nucleotide, G530, stacks with the side chain of SmpB residue His136, inducing conformational changes that lead to GTP hydrolysis by EF-Tu. A portion of the C-terminal tail forms a helix within the mRNA channel, monitoring the length of mRNA bound in the ribosome to avoid aborting productive protein synthesis. Helix formation in the mRNA channel is essential for accommodation and peptidyl transfer, but not for GTP hydrolysis. We show that conserved residues in the tail are essential for EF-Tu activation, accommodation, or translocation to the P site. Our findings lead to a clearer model of how the tmRNA-SmpB complex enters stalled ribosomes.

<p>Ribosomes stalled on problematic mRNAs can be rescued by a mechanism called <i>trans</i>-translation. This mechanism employs a dual transfer-messenger RNA molecule (tmRNA) together with a helper protein (SmpB). </p><p>In this work we have used an <i>in vitro</i> translation system with pure components to further clarify the roles of tmRNA and SmpB in <i>trans-</i>translation. </p><p>We found that SmpB binds ribosomes <i>in vivo</i> and <i>in vitro</i> independently of tmRNA presence and is essential for tmRNA binding and <i>trans-</i>peptidation. We show that two SmpB molecules can bind per ribosome, that SmpB does not leave the ribosome after <i>trans-</i>peptidation and that SmpB pre-bound to the ribosome can trigger <i>trans-</i>translation. </p><p>We demonstrated that the rate of <i>trans-</i>transfer of a peptide from the P-site tRNA to Ala-tmRNA and the efficiency by which Ala-tmRNA competes with peptide release factors decrease with increasing the mRNA length downstream from the P site of the ribosome. We showed that <i>trans-</i>translation is strongly stimulated by RelE cleavage of A-site mRNA. We concluded that tmRNA action<i> in vivo</i> must always be preceded by mRNA truncation.</p><p>We showed that rapid release of truncated mRNAs from the ribosome requires translocation of the peptidyl-tmRNA into the ribosomal P site, which is strictly EF-G dependent. mRNA release is slowed down by strong Shine and Dalgarno like sequences upstream the A site and by long 3’-extensions downstream from the P-site codon. </p><p>Footprinting was used to monitor SmpB binding to tmRNA, ribosomes and subunits and to study tmRNA interactions with the ribosome at distinct <i>trans-</i>translation stages. We confirmed that two SmpB molecules bind per ribosome and interact with nucleotides below the L7/L12-stalk on the 50S subunit and near the subunit interface on the 30S. We showed that tmRNA is mostly in complex with SmpB <i>in vivo</i> and during <i>trans-</i>translation. Specific cleavage patterns of tmRNA were observed at different stages of <i>trans-</i>translation, but the overall tmRNA conformation seems to be maintained during the whole process.</p>

Ribosomes stalled on problematic mRNAs can be rescued by a mechanism called trans-translation. This mechanism employs a dual transfer-messenger RNA molecule (tmRNA) together with a helper protein (SmpB). In this work we have used an in vitro translation system with pure components to further clarify the roles of tmRNA and SmpB in trans-translation. We found that SmpB binds ribosomes in vivo and in vitro independently of tmRNA presence and is essential for tmRNA binding and trans-peptidation. We show that two SmpB molecules can bind per ribosome, that SmpB does not leave the ribosome after trans-peptidation and that SmpB pre-bound to the ribosome can trigger trans-translation. We demonstrated that the rate of trans-transfer of a peptide from the P-site tRNA to Ala-tmRNA and the efficiency by which Ala-tmRNA competes with peptide release factors decrease with increasing the mRNA length downstream from the P site of the ribosome. We showed that trans-translation is strongly stimulated by RelE cleavage of A-site mRNA. We concluded that tmRNA action in vivo must always be preceded by mRNA truncation. We showed that rapid release of truncated mRNAs from the ribosome requires translocation of the peptidyl-tmRNA into the ribosomal P site, which is strictly EF-G dependent. mRNA release is slowed down by strong Shine and Dalgarno like sequences upstream the A site and by long 3’-extensions downstream from the P-site codon. Footprinting was used to monitor SmpB binding to tmRNA, ribosomes and subunits and to study tmRNA interactions with the ribosome at distinct trans-translation stages. We confirmed that two SmpB molecules bind per ribosome and interact with nucleotides below the L7/L12-stalk on the 50S subunit and near the subunit interface on the 30S. We showed that tmRNA is mostly in complex with SmpB in vivo and during trans-translation. Specific cleavage patterns of tmRNA were observed at different stages of trans-translation, but the overall tmRNA conformation seems to be maintained during the whole process.

<p>5-azacytidine and its derivatives are cytidine analogs used for leukemia chemotherapy. The primary effect of 5-azacytidine is the prohibition of cytosine methylation, which results in covalent DNA-methyltransferase crosslinks at cytosine methylation sites. These DNA-protein crosslinks have been suggested to cause chromosomal rearrangements and contribute to cytotoxicity, but the detailed mechanisms of DNA damage and the repair pathways of DNA-protein crosslinks have not been elucidated. </p><p>We used 2-dimensional agarose gel electrophoresis and electron microscopy to analyze plasmid pBR322 replication dynamics in Escherichia coli cells grown in the presence of 5-azacytidine. 2-dimensional gel analysis revealed the accumulation of specific bubble- and Y-molecules, dependent on overproduction of the cytosine methyltransferase EcoRII and treatment with 5-azacytidine. Furthermore, a point mutation that eliminates a particular EcoRII methylation site resulted in disappearance of the corresponding bubble- and Y-molecules. These results imply that 5-azacytidine-induced DNA-protein crosslinks block DNA replication in vivo. RecA-dependent X-structures were also observed after 5-azacytidine treatment. These molecules may be generated from blocked forks by recombinational repair and/or replication fork regression. In addition, electron microscopy analysis revealed both bubbles and rolling circles after 5-azacytidine treatment. These results suggest that replication can switch from theta to rolling circle mode after a replication fork is stalled by a DNA-methyltransferase crosslink. The simplest model for the conversion of theta to rolling-circle mode is that the blocked replication fork is cleaved by a branch-specific endonuclease. Such replication-dependent DNA breaks may represent an important pathway that contributes to genome rearrangement and/or cytotoxicity. </p><p>In addition, we performed a transposon mutagenesis screen and found that mutants defective in the tmRNA translational quality control system are hypersensitive to 5-azacytidine. The hypersensitivity of these mutants requires expression of active methyltransferase, indicating that hypersensitivity is dependent on DNA-methyltransferase crosslink formation. Furthermore, the tmRNA pathway is activated upon 5-azacytidine treatment in cells expressing methyltransferase, resulting in increased SsrA tagging of cellular proteins. These results support a "chain-reaction" model, in which transcription complexes blocked by 5-azacytidine-induced DNA-protein crosslinks result in ribosomes stalling on the attached nascent transcripts, and the tmRNA pathway is invoked for cleaning up the resulting pile-ups. In support of this model, an ssrA mutant is also hypersensitive to antibiotic streptolydigin, which blocks RNA polymerase elongation. These results reveal a novel role for the tmRNA system in clearance of coupled transcription/translation complexes in which RNA polymerase has become blocked.</p> / Dissertation

In eubacteria, stalled ribosomes are rescued by a conserved quality-control mechanism involving transfer-messenger RNA (tmRNA) and its protein partner SmpB. Mimicking a tRNA, tmRNA enters stalled ribosomes, adds Ala to the nascent polypeptide, and serves as a template to encode a short peptide that tags the nascent protein for destruction. To further characterize the tagging process, we developed two genetic selections that link tmRNA activity to cell death. These negative selections can be used to identify inhibitors of tagging or to identify mutations in key residues essential for ribosome rescue. Little is known about which ribosomal elements are specifically required for tmRNA activity. Using these selections, we isolated ribosomal RNA mutations that block the rescue of ribosomes stalled at rare Arg codons or at the inefficient termination signal Pro-opal. We find that deletion of A1150 in the 16S rRNA blocks tagging regardless of the stalling sequence, suggesting that it inhibits tmRNA activity directly. The C889U mutation in 23S rRNA, however, lowers tagging levels at Pro-opal and rare Arg codons but not at the 3'-end of an mRNA lacking a stop codon. We conclude that the C889U mutation does not inhibit tmRNA activity per se but interferes with an upstream step intermediate between stalling and tagging.

The work discussed in this thesis deals with the significance of initiator tRNA gene copy number in Escherichia coli. A summary of the relevant literature discussing the process of protein synthesis, initiator tRNA selection and gene redundancy is presented in Chapter 1. Chapter 2 describes the ‘Materials and Methods’ used in the experimental work carried out in this thesis. The next three chapters address the significance of initiator tRNA gene copy number in E. coli at three levels; at the level of the molecule (Chapter 3), at the level of the cell (Chapter 4) and at the level of the population (Chapter 5). At the end of the thesis are appended three publications, which include two papers where I have contributed to work not discussed in this thesis and one review article. A brief summary of chapters 3 to 5 is provided below: (i) Chapter 3: Can E. coli remain viable without the 3 G-C base pairs in initiator tRNA? Initiator tRNAs are distinguished from elongator tRNAs by several features key among which are the three consecutive and near universally conserved G-C base pairs found in the anticodon stem of initiator tRNAs. These bases have long been believed to be essential for the functioning of a living cell, both from in vitro and in vivo analysis. In this study, using targeted mutagenesis and an in vivo genetics based approach, we have shown that the 3 G-C base pairs can be dispensed with in E. coli, and the cell can be sustained on unconventional initiator tRNAs lacking the intact 3 G-C base pairs. Our study uncovered the importance of considering the relative amounts of molecules in a living cell, and their role in maintaining the fidelity of protein synthesis. (ii) Chapter 4: Can elongator tRNAs initiate protein synthesis? There are two types of tRNAs; initiator tRNA, of which there is one representative in the cell, and elongator tRNAs of which there are several representatives. In this study, we have uncovered initiation of protein synthesis by elongator tRNAs by depleting the initiator tRNA content in the cell. This raises the possibility that competition between initiator and elongator tRNAs at the P site of the ribosome occurs routinely in the living cell, and provides a basis for initiation at several 'start' sites in the genome that may not be currently annotated as such. We speculate that such a phenomenon could be exploited by the cell to generate phenotypic diversity without compromising genomic integrity. (iii) Chapter 5: How many initiator tRNA genes does E. coli need? E. coli has four genes that encode initiator tRNA, these are the metZWV genes that occur at 63.5 min in the genome, and the metY gene that occurs at 71.5 min in the genome. Earlier studies indicated that the absence of metY had no apparent impact on cell growth. In view of the importance of initiator tRNA gene copy number in maintaining the rate and fidelity of protein synthesis, we examined the fitness of strains carrying different numbers of initiator tRNA genes by competing them against each other in both rich and limited nutrient environments. Our results indicate a link between caloric restriction and protein synthesis mediated by the initiator tRNA gene copy number.