1,721,118 research outputs found
A Study On The Mechanism Of Initiator tRNA Selection On The Ribosomes During Translation Initiation And Rescue Of The Stalled Ribosomes By SsrA In Escherichia Coli
Get PDFThe studies reported in this thesis describe the work done in the area of translation initiation where a previously unknown role of multiple copies of initiator tRNA in E. coli has been reported. Also the role of SsrA resume codon in resumption of translation, until not clearly known has been reported here. Chapter -1 discusses the relevant literature in understanding translation and initiator tRNA selection on the ribosome during initiation. It also discusses the literature pertaining to the aspect of release of stalled ribosomal complexes by SsrA. This is followed by the next chapter (chapter- 2) which discusses the materials and methods used throughout the study. Chapter- 3 describes the studies leading to the role of multiple copies of initiator tRNA in E. coli in governing the fidelity of initiator tRNA selection on the P site of the ribosome. This is followed by Chapter-4 which describes the role of the resume codon of the SsrA in governing the efficiency of trans-translation in releasing the stalled ribosomal complexes. The summaries of the chapters 3 and chapter 4 are briefly described below.
i) Role of conserved 3GC base pairs of initiator tRNA in the initiator-elongator tRNA discrimination.
Translation initiation is the first step in the very important and highly conserved biological process of protein biosynthesis. The process involves many steps, a wide array of protein factors at each specialized step and a large ribonucleoprotein particle; the ribosome to decode the information of the mRNA template into biologically active proteins. The process of initiation is still unclear largely due to fewer reports of available structural data. One of the very interesting questions that people have been trying to address is how the initiator tRNA is selected on the P- site of the ribosome and what is the importance of the conserved three GC base pairs in the anticodon stem of the initiator tRNA. Here in this study, I have studied this question by using the classical genetic technique of generating and characterizing the mutant initiator tRNA defective at the step of initiation. I have identified and analyzed the suppressors which are capable of rescuing this defect in initiation. The study involves two such E. coli suppressor strains (named D4 and D27). These suppressors can initiate translation from a reporter CAT mRNA with amber codon, independent of the presence of the three consecutive GC base pairs in the anticodon stem of initiator tRNAs. Mapping of the mutations revealed that the mutants are defective in expression of the tRNA1fMet (metZVW) gene locus which encodes the initiator tRNA. Both the suppressors (D4 and D27) also allow initiation with elongator tRNA species in E. coli. Taken together, the results show that E. coli when deficient in the initiator tRNA concentration can lead to initiation with elongator tRNA species.
ii) The Role of SsrA/tmRNA in ribosome recycling and rescue.
Occasionally during the process of translation, the ribosomes stall on the mRNA before the polypeptide synthesis is complete. This situation is detrimental to the organism because of the sequestration of the tRNAs as ‘peptidyl tRNAs’ and the ribosomes. In E. coli one of the pathways to rescue stalled ribosomes involves disassembly of these stalled complexes to release peptidyl tRNAs which are then recycled by peptidyl tRNA hydrolase (Pth), an essiential enzyme in E. coli. The other pathway which is not essential in E. coli but is conserved in all prokaryotes involves SsrA or tmRNA (transfer messenger RNA). The tmRNA is charged with alanine and recognizes the stalled ribosomal complexes and acts as tRNA to bind the A-site. It also functions as mRNA by adding a undecapeptide (which is actually a tag for degradation by cellular proteases) to the existing polypeptide and there is normal resumption of the translation. In most sequences of SsrA ORF, the first codon of the ORF, called as resume codon, is conserved. I wanted to understand the importance of the conservation of the resume codon. Towards this end I randomly mutated the resume codon and studied the effect of the altered resume codon in the rescue of stalled ribosomal complexes. The effect of over-expression of these mutants was investigated in the rescue of the Pthts defect since it is known that the overexpression of SsrA rescues the temperature sensitive phenotype of the Pthts strain and so causes less accumulation of peptidyl–tRNA in E. coli .The effect for these mutants has also been studied by the growth of hybrid λimmP22 phages. I also used AGA minigene system to study the effect of various mutants which has been shown to sequester tRNAArg (UCU) in the ribosomal P-site, translation of this minigene causes toxicity to E. coli. I have tried to study the effect of the SsrA mutants in rescue of toxicity caused by the minigene. Overall, the observations indicate that the conservation of the resume codon is important in E. coli and having mutated resume codon probably leads to deficient trans-translation during one or the other growth conditions
Roles of the ribosomal protein uS12 and the initiation factor 3 in the maintenance of fidelity of translation in Escherichia coli
No full textThe flow of genetic information within biological systems according to the central dogma of molecular biology entails protein synthesis as the last step towards deciphering the message encrypted in the genetic code in cells. Translation occurs in four distinct steps: initiation, elongation, termination and ribosome recycling. To ensure faithful translation, the macromolecular protein synthesizing machinery, the ribosome, employs several check-points monitored by the ribosomal RNAs, ribosomal proteins and the translation factors. The ribosomal protein uS12 has been studied extensively for its role in maintaining the fidelity of translation elongation in bacteria and eukaryotes. The overarching highlight of the study is identification of novel roles played by the ribosomal protein uS12 in Escherichia coli in translation at and beyond the step of elongation owing to its unique molecular location in the ribosome. The work has revealed novel aspects of interplay between the initiator tRNA, initiation factor 3 (IF3) and uS12 in ensuring the fidelity of translation initiation. Furthermore, I made use of the E. coli model system to uncover many new functions of the PNSA loop of uS12 which had, till now, exclusively been studied for its effects on maintenance of the fidelity of translation elongation.
The thesis comprises five major chapters and two appendices. Chapters 1 and 2 provide relevant details of the literature review (Chapter 1) and materials and methods (Chapter 2). The major research findings of the study are described in the subsequent chapters. Summaries of these works (Chapters 3-5 and, Appendices 1 and 2) are provided as follows.
(A) Co-occurrence of mutations in initiation factor 3 and the ribosomal protein uS12 rescues slow growth phenotype of Escherichia coli sustained on an initiator tRNA with a mutant anticodon stem (Chapters 3 and 4)
Initiator tRNAs (i-tRNAs) are special molecules possessing a highly conserved sequence of three consecutive GC base pairs (3GC pairs or GC/GC/GC) at 29:41, 30:40, and 31:39 positions in the anticodon stem which distinguishes them from the pool of elongator tRNAs. In E. coli, the 3GC pairs are known to target the i-tRNA to the ribosomal P-site, assist transiting the i-tRNA through various phases of initiation and play an crucial role in the ultimate steps of 16S rRNA maturation. To understand the role of the full complement of 3GC pairs in i-tRNAs, we used genetic methods to isolate fast growing suppressors of an E. coli strain sustained on i-tRNA having cg/GC/cg sequence instead of GC/GC/GC. Characterization of the suppressor strains revealed that the compensatory mutations are found in the infC gene that codes for IF3 and in the rpsL gene encoding uS12. The two suppressors that have been characterised (Sup-1 and Sup-2) each had the common mutation of V93A in IF3 (infC), and while the Sup-1 had an additional mutation of V32L in uS12 (rpsL), Sup-2 possessed H76L mutation in uS12. Detailed analyses reveal that the V93A mutation in IF3 was already present in the strain and it led to relaxed fidelity of i-tRNA selection to allow initiation with i-tRNA having cg/GC/cg (in place of the highly conserved sequence of GC/GC/GC) and the slow growth phenotype of the strain. Rescue of growth occurred by additional mutations in uS12 (in Sup-1 and Sup-2) which allowed improved fidelity of i-tRNA selection at the step of initiation. We show that the H76L mutation in uS12 in Sup-2 conferred better fidelity at the step of i-tRNA selection than did the V32L mutation in Sup-1. Importantly, the V32L mutation (Sup-1) compensated for the deficiency of fidelity of i-tRNA selection by ensuring an efficient dissociation of the 70S initiation complexes or the ribosomes stalled during elongation. Thus, our study highlights the allele-specific evolution of the mutations in IF3 and uS12 to salvage the retarded growth due to an i-tRNA containing cg/GC/cg sequence. The adaptive evolution in IF3 followed by the directional selection of the mutation in uS12, provide us with the opportunity to comment on the sequence of co-evolution of the translation apparatus in certain mycoplasma. Genetic and biochemical data presented establish the crucial role of uS12 in translation initiation and recycling. We describe unique genetic networks between uS12, IF3 and i-tRNA in initiation and between uS12, Pth (peptidyl-tRNA hydrolase), EF-G (elongation factor-G) and RRF (ribosome recycling factor) in recycling which, taken together, can govern the fidelity of initiation of translation in bacteria.
(B) A mutation in the ribosomal protein uS12 reveals novel functions of its universally conserved PNSA loop (Chapter 5)
The small ribosomal protein, uS12 has classically been studied for its importance in fidelity of translation elongation. The 44PNSA47 and 90PGVR93 loops of the protein (proximal to the codon:anticodon helix at the ribosomal A-site) are conserved across all domains of life. A recent article on mutations in uS12 associated with a human ribosomopathy led us to investigate the roles of the same mutations in a haploid genome system of E. coli, considering that effects of such mutations may be observed in a total context as opposed to partial effects in heterozygous systems. The unique location of uS12 at the inter-subunit surface and at the A-site of the 30S of bacterial ribosomes, has led us to uncover other functions of the PNSA loop by analysis of a mutation in the PNSA loop. We show that apart from its well-established functions, the PNSA loop is also involved in the fidelity of translation initiation, ribosome recycling and ribosome biogenesis. We also establish genetic interactions of the protein uS12 with Peptidyl-tRNA hydrolase (Pth) and affirm that interactions with the ribosome recycling factor (RRF). Our data also suggest that the role of AUU initiation codon in IF3 goes beyond its autoregulation and may have global effects on translation.
(C). Studies using Drosophila: The other studies that I carried out during the course of my research are attached as appendices as upon their completion, they do not necessarily adhere with the theme of maintenance of the fidelity of translation.
Appendix 1. Identification of a putative fertility factor in Drosophila melanogaster.
It has been shown in E. coli that lower amounts of i-tRNA may play a role in survival under nutrient stress conditions. Having a lower amount of initiator tRNA and hence, a lower rate of protein synthesis, provides an advantage in growth under such situations. We chose to work on Drosophila melanogaster as our model system to study the role of i-tRNAs during development. It is a holometabolous insect with four stages in its life cycle, namely, egg, larva, pupa and adult in said order; and for its development, the different stages require a different set of proteins. The i-tRNAs are important as they are one of the key participants in initiation of translation (which is the rate limiting step in translation) leading to the necessary change of proteome during development. We studied a fly line homozygous for insertion of an extra copy of initiator tRNA gene in each of its third chromosome and soon discovered that the male flies were rendered sterile. By genetic experiments we ruled out the involvement of the i-tRNA in the sterility phenotype and confirmed the cause to be random insertion of the P-element carrying the extra copy of i-tRNA gene. The exact molecular localization of the P-element contributing to fertility of the fruit fly is being investigated.
Appendix 2. Development of mCherry tagged UdgX as a highly sensitive molecular probe for specific detection of uracils in DNA
Presence of uracil in DNA has been implicated in the development of Drosophila melanogaster, life cycle of Plasmodium falciparum, and antibody maturation in B lymphocytes in animalia. However, the field lacked a facile, robust and highly specific method to detect uracil in DNA especially for in situ analysis. The novel mycobacterial protein, MsmUdgX, identified and characterized from our laboratory, forms an unusually tight (covalent) complex with uracil containing DNA in single or double stranded forms in a highly specific manner. This study describes the use of mCherry tagged MsmUdgX (mChUdgX) to combine the property of UdgX to tightly and specifically bind to the uracil sites in the genome, with the sensitivity of fluorescent detection of mCherry as a sensor. We show that both the purified mChUdgX as well as E. coli cell extracts over-expressing the chimeric protein provide high sensitivity of detecting uracil in DNA with high specificity. The novelty of the assay developed lies in the simplicity in preparation of the sensor. The technique described minimizes tedious sample preparation protocols, does not depend on antibody-based detection, is quicker and can be applied to visualize uracil incorporation in varied contexts
DNA Repair Proteins in Mycobacteria and their Physiological Importance
Get PDFDNA repair proteins in mycobacteria and their physiological importance
Mycobacterium tuberculosis, the causative organism of tuberculosis, resides in the host macrophages where it is subjected to a plethora of stresses like reactive oxygen species (ROS) and reactive nitrogen intermediate(RNI) which are generated as a part of the host’s primary immune response. These stresses can damage the cellular components of the pathogen including DNA and its precursors. Two common damages to DNA and its precursors caused by ROS and RNI are oxidation of guanine to 8-oxo-guanine and deamination of cytosine to uracil. Mycobacteria, which are known to have high G+C content, must be more susceptible to such damages, and are thus equipped with the mechanisms to counteract these damages. One such mechanism is to hydrolyse the 8-oxo-dGTP into 8-oxo-dGMP to avoid its incorporation in the DNA during its synthesis. This job is done by a protein called MutT.In mycobacteria four homologs of MutT, namely MutT1, MutT2, MutT3 and MutT4 have been annotated. The second mechanism deals with the repair of uracil residues present in DNA which are generated by deamination of cytosines or incorporation of dUTP during DNA synthesis. This is taken care of by a protein called uracil DNA glycosylase (UDG) which excises uracil by cleaving the N-C1’ glycosidic bond between the uracil and the deoxyribose sugar in a DNA repair pathway called the base excision repair (BER). In this study, the biochemical properties and physiological role of mycobacterial MutT2 and, MSMEG_0265 (MsmUdgX), a novel uracil DNA glycosylase superfamily protein, have been investigated.
I.Biochemical characterization of MutT2 from mycobacteria and its antimutator role.
Nucleotide pool, the substrate for DNA synthesis is one of the targets of ROS which is generated in the macrophage upon Mycobacterium tuberculosis infection. Thus, the pathogen is at increased risk of accumulating oxidised guanine nucleotides such as 8-oxo-dGTP and 8-oxo-GTP. By hydrolysing the damaged guanine nucleotides before their incorporation into nucleic acids, MutT proteins play a critical role inallowing organisms to avoid their deleterious effects. Mycobacteria possess several MutT proteins. Here, we have purified recombinantM. tuberculosisMutT2 (MtuMutT2) andM. smegmatisMutT2 (MsmMutT2) proteins as representative of slow and fast growing mycobacteria, for the purpose of biochemical characterization. UnlikeEscherichia coliMutT, which hydrolyzes 8-oxo-dGTP and 8-oxo-GTP, the mycobacterial proteins hydrolyze not only 8-oxo-dGTP and 8-oxo-GTP but also dCTP and 5-methyl-dCTP. Determination of kinetic parameters (KmandVmax) revealed thatwhileMtuMutT2 hydrolyzes dCTP nearly four times better than it does 8-oxo-dGTP,MsmMutT2 hydrolyzes them almost equally well. Also,MsmMutT2 is about 14 times more efficient thanMtuMutT2 in its catalytic activity of hydrolyzing 8-oxo-dGTP.Consistent with these observations,MsmMutT2 but notMtuMutT2 rescuesE. colifor MutT deficiency by decreasing both themutation frequency and A to C mutations (a hallmark of MutT deficiency). We discuss these findings in the context of the physiological significance of MutT proteins.
II.Understanding the biochemical properties of MSMEG_0265 (MsmUdgX), a novel uracil DNA glycosylase superfamily protein
Uracil DNA glycosylases (UDGs) are base excision repair enzymes which excise uracil from DNA by cleaving the N-glycosidic bond. UDGs are classified into 6 different families based on their two functional motifs, i. e.,motif A and motif B. In mycobacteria, there are two uracil DNA glycosylases, Ung and UdgB which belong to Family 1 and Family 5, respectively. In this study, based on the presence of the two functional motifs, we have discovered yet another uracil DNA glycosylase in M. smegmatis, which we have called MsmUdgX.The motif A and motif B of this protein indicate that it does not belong to any of the UDG families already classified but has highest similarity with Family 4 UDGs. Homologs of this protein are also present in several other organisms like M. avium, Streptomyces ceolicolor, Rhodococcus etc., but absent in M. tuberculosis, archaea and eukaryotes. Activity assays of this protein show that unlike other UDGs, MsmUdgX does not excise uracil, but forms a tight complex with uracil containing single stranded (ss) and double stranded (ds) DNAs, as observed by a shifted band in 8M urea-PAGE as well as SDS-PAGE. It also does not recognize other modified nucleotides that we investigated, in DNA. The protein binds to uracil-DNA in a wide range of pH and the minimum substrate required for its binding is pNUNN. Like Family 4 UDG, the protein has Fe-S cluster but it is not as thermostable as the Family 4 UDGs. Addition of different metal ions does not affect its binding property, and even the presence of M. smegmatis cell free extract does not diminish its binding activity. Since this protein binds specifically to uracil in DNA, an application of the protein for detection of uracil in the genomic DNA is proposed.
III. Elucidation of the role of KRRIH loop in MsmUdgX by mutational analysis
MsmUdgX is a novel uracil DNA glycosylase superfamily protein which has the highest homology to Family 4 UDGs. However, alignment of MsmUdgX amino acid sequence with that of Family 4 UDGs shows that there is an extra stretch of amino acids which is unique to this group of proteins. This stretch, defined by AGGKRRIH is absent in all Family 4 UDGs and the region KRRIH of the strtch is quite conserved amongst all UdgX proteins. Homology modelling of MsmUdgX, using a Family 4 UDG (TthUdgA) shows that this extra stretch of amino acids forms an outloop near the enzyme active site. Another unique difference between MsmUdgX and Family 4 UDGs is in the motif A where MsmUdgX has GEQPG and the Family 4 UDGs haveGE(A/G)PG. Our work on MsmUdgX has shown that, unlike other UDGs, this protein does not excise uracils, but forms a tight complex with the uracil containing DNA. This unique tight uracil binding property as well as KRRIH amino acid stretch has not been observed for any uracil DNA glycosylase superfamily proteins. So, to gain insight into the role of KRRIH and glutamine (Q) of motif A in MsmUdgX family of proteins, site directed mutagenesis was done in this region and we observed that mutation of His109 of the KRRIH loop to serine (S) leads to a gain of uracil excision activity, whereas changing the R107 to S, ‘RRIH’ to ‘SSAS’ or deleting the loop altogether leads to loss of its complex formation activity. Further, mutation of H109 to other amino acids like G, Q and A also shows uracil excision activity. Mutation of the glutamine in the motif A to alanine so that it is exactly similar to that of Family 4 UDGs, does not affect its uracil binding activity. This observation indicates that the KRRIH loop has an important role in the tight binding and/or uracil excision activity of MsmUdgX. Crystal structure of MsmUdgX in complex with uracil-DNA oligo and MsmUdgX H109S mutants are being studied.IV.
Physiological importance of MsmUdgX in M. smegmatis
MsmUdgX is a uracil DNA glycosylase superfamily protein which binds tightly to uracil (in DNA) without excising it. To elucidate its role in M. smegmatis, knockout of udgX was generated. Growth comparison of the wild type and the ΔudgX strains does not show any growth differences under the conditions tested. However, overexpression of MsmUdgX in recA deficient strains of E. coli as well as M. smegmatis leads to their retarded growth. Retarded grown is also observed in strains deficient in other DNA repair proteins that work in conjunction with RecA. These observations indicate that repair/release of MsmUdgX-uracil DNA complex might be a RecA dependent process
Metabolism Of Queuosine, A Modified Nucleoside, In Escherichia Coli And Caenorhabditis Elegans And Dual Function Of Bovine Mitochondrial Initiation Factor 2 As Initiation Factors 1 And 2 In Escherichia Coli
No full textThe studies reported in this thesis address firstly, the biology of a modified nucleoside, Queuosine (Q) and secondly, the properties of mitochondrial translation initiation factor 2. A summary of the relevant literature on both these topics is presented in Chapter 1. Section I of this ‘General Introduction’ summarizes the literature on biosynthesis and physiological importance of Queuosine. Section II is a brief review of the current understanding of translation initiation in Eubacteria. Information about the mitochondrial translation initiation apparatus also features as a subsection. The next chapter (Chapter 2), describes the ‘Materials and Methods’ used throughout the experimental work presented in the thesis. It is followed by three chapters containing experimental work as described below:-
i) Biosynthesis of Queuosine (Q) in Escherichia coli
Q is a hypermodification of guanosine found at the wobble position of tRNAs with GUN anticodons. Q is thought to be produced via a complex multistep pathway, the details of which are not known. It was found in our laboratory that a naturally occurring strain of E. coli B105 lacked Q modification in the tRNAs. As the known enzymes of Q biosynthesis were functional in this strain, it presented us with the opportunity to uncover novel component(s) of Q biosynthetic pathway. In the present work, a genetic screen was developed to map the defect in E. coli B105 to a previously uncharacterised gene, ybaX, predicted to code for a 231 amino acid long protein with a pI of 5.6. Further genetic analyses showed that YbaX functions at a step leading to production of preQ0, the first known intermediate in the generally accepted pathway that utilizes GTP as the starting molecule. The gene ybaX has been renamed as queC. Using a combination of bioinformatics based prediction and gene knockouts, we have also been able to place two more genes, queD and queE at the initial step in Q biosynthesis, suggesting that the initial reaction of Q biosynthesis might be more complex and mechanistically different than what has been proposed earlier.
ii) Caenorhabditis elegans as a Model System to Study Queuosine Metabolism in Metazoa
Animals are thought to obtain Q (or its analogs) as a micronutrient from dietary sources such as gut microflora, and the corresponding base is then inserted in the substrate tRNAs by tRNA guanine transglycosylase (TGT). In animal cells, changes in the abundance of Q have been shown to correlate with diverse phenomena including stress tolerance, cell proliferation and tumor growth but the precise function of Q in animal tRNAs remains unknown. A major obstacle in the study of Q metabolism in higher organisms has been the requirement of a chemically defined medium to cause Q depletion in animals. Having discovered that E. coli B105 has a block in the initial step of Q biosynthesis, we reasoned that this strain could be used as a Q- diet for organisms like C. elegans, which naturally feed on bacteria. An analysis of C. elegans tRNA revealed that as in the other higher animals, tRNAs in the worm C. elegans, are modified by Q and its sugar derivatives. When the worms were fed on Q deficient E. coli B105, Q modification was absent from the worm tRNAs suggesting that C. elegans lacks a de novo pathway of Q biosynthesis. The inherent advantages of C. elegans as a model organism, the speed and simplicity of conferring a Q deficient phenotype on it, make it an ideal system to investigate the function of Q modification in tRNA. By microinjecting tgt-1-gfp constructs into C. elegans, we could also demonstrate that a major form of TGT is localised to the nucleus, suggesting that insertion of Q into the tRNAs could be occurring in the nucleus.
iii) Dual Function of Bovine Mitochondrial Initiation Factor 2 as Initiation Factors 1 and 2 in Escherichia coli
Translation initiation factors 1 and 2 (IF1 and IF2) are known as ‘universal translation initiation factors’ due to the presence of their homologs in all living organisms. Homologs of these factors are also present in the chloroplast, however, a unique situation exists in the mitochondria where IF2 homolog (IF2mt) is known to occur but an IF1 like factor is not found. We have engineered a system of E. coli knockouts to allow the study of IF2mt in a prokaryotic milieu. We found that the bovine IF2mt complements an E. coli strain wherein the gene for IF2 is knocked out, providing the first proof of a mitochondrial translation initiation factor working in a eubacterial system. This conservation of function is especially interesting in light of the recent reports revealing significant differences between the mitochondrial and eubacterial ribosomes. Further, we found that the IF2mt can also support a double knockout of IF1 and IF2 genes in E. coli, suggesting that IF2mt possesses both IF1 and IF2 like activities in E. coli. This finding offers an explanation for the lack of an IF1 like factor in mitochondria. Molecular modeling of bovine IF2mt indicated that a conserved insertion found in all mitochondrial IF2s, may form a protruding α-helix that could stabilize IF2mt on ribosomes. This insertion could in principle function as IF1 and we have explored the role of this conserved insertion both in vivo and in vitro, by generating mutants of IF2mt and EcoIF2, to lose or gain the conserved insertion respectively
Mechanism of Recycling of Ribosomes Stalled on mRNAs in Escherichia Coli
Get PDFStudies reported in this thesis address the question of how pre-termination ribosomal complexes stalled during translation of mRNA are recycled. The process of recycling of the stalled ribosomes involves many translational factors. During the course of my studies, I have uncovered new roles of SsrA (tmRNA), IF3 and ribosome recycling factor (RRF) in recycling stalled ribosomes. These findings are summarized as follows:
(i) A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in
Escherichia coli
The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions. The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on mRNAs and targets the aberrant protein for degradation. In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs. The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide. As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed. Thus, we hypothesized that tmRNA may rescue a defect in Pth. The findings of the experiments detailed in this thesis show that SsrA rescues a defect in Pth by reducing the peptidyl-tRNA load on Pth.
(ii) Evidence for a role of initiation factor 3 in recycling ribosomal complexes stalled on mRNAs in Escherichia coli.
Specific interactions between ribosome recycling factor (RRF) and EF-G mediate disassembly of post-termination ribosomal complexes for new rounds of initiation. The
interactions between RRF and EF-G are also important in peptidyl-tRNA release from pre-termination complexes. Unlike the post-termination complexes (harboring tRNA), the pre-termination complexes (harboring peptidyl-tRNA) are not recycled by RRF and EF-G in vitro, suggesting participation of additional factor(s) in the process. Using a combination of biochemical and genetic approaches, we show that, 1. Inclusion of IF3 with RRF and EF-G results in recycling of the pre-termination complexes; 2. IF3 overexpression in Escherichia coli LJ14 rescues its temperature sensitive phenotype for RRF; (3) Transduction of infC135 (encoding functionally compromised IF3) in E. coli LJ14 generates a ‘synthetic severe’ phenotype; (4) The infC135 and frr1 (a promoter down RRF gene) alleles synergistically rescue a temperature sensitive mutation in peptidyl-tRNA hydrolase in E. coli; and (5) IF3 facilitates ribosome recycling by Thermus thermophilus RRF and E. coli EFG in vivo and in vitro. These lines of evidence clearly demonstrate the physiological importance of IF3 in the overall mechanism of ribosome recycling in E. coli.
(iii) The role of RRF in dissociating of pre-termination ribosomal complexes stalled during elongation
Translating ribosomes often stall during the repetitive steps of elongation for various reasons. The stalled ribosomes are rescued by the process of trans-translation involving tmRNA (SsrA) or by a factor mediated dissociation of the stalled ribosome into its subunits leading to the drop-off of the peptidyl-tRNA. The mechanistic details of how the factor mediated dissociation is carried out, is not well studied. Studies described in the above section have highlighted the role of RRF in dissociating stalled pre-termination complexes. However, the in vivo studies in this area have been limited for lack of defined pre-termination complexes. Two in vivo systems based on translation of AGA minigene and the ung gene (EcoUngstopless) transcripts were designed. Evidence is presented to show that translation of both of these transcripts is toxic to E. coli because of the accumulation of the transcript specific stalled pre-termination complexes. Availability of these model systems has allowed us to address the role of RRF in dissociating stalled ribosomes. We show that RRF rescues stalled ribosomes on these constructs and its overexpression can rescue the toxicity. The physiological importance of this observation is highlighted by the rescue of AGA minigene inhibitory effect on λimmP22 hybrid phage growth upon RRF overexpression
How Much Initiator tRNA Does Escherichia Coli Need?
Get PDFThe 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
The Role of Initiation Factor 3 : Insights from E. Coli, Mitochondria and Mycoplasma
Get PDFThe process of translation initiation is the most highly regulated step of protein synthesis. In bacteria, three initiation factors (IF1, IF2 and IF3) play crucial roles during initiation. IF3 acts as an anti-association factor for the two ribosomal subunits. Eubacterial IF3 also permits initiator tRNA (i-tRNA) selection at the P site of the ribosome. Two features of i-tRNA, i. e. the characteristic 3GC base pairs in the anticodon stem and the cognate interaction of the anticodon sequence with the initiation codon of the mRNA contribute to IF3 based selection and/or proofreading. However, the exact mechanism of this discrimination and the contribution of the individual domains towards this process of selection/ proofreading are unclear. Further, there are exceptional instances in the natural world where either the codon-anticodon interaction or the anticodon stem composition deviates from the norm. For instance, in mammalian mitochondria, non-AUG codons such as AUU and AUA are present in the genome although they are notoriously poor initiation codons. In addition, some species of Mycoplasma have i-tRNAs with variations in the typically conserved 3GC base pairs of the anticodon stem. In this study, we have investigated the mechanism of proofreading activity of IF3 of E. coli, mitochondrial and mycoplasmal origins.
Part I: Proofreading function of IF3 in E. coli
IF3 is composed of N and C terminal domains joined by a flexible linker region. By means of complete and partial IF3 knockouts, we show that the C-terminal domain (CTD) is essential for the survival of E. coli while the N-terminal (NTD) is required for cellular fitness. Using reporter assays, we have established the role of the NTD in proofreading, while polysome profile analyses reaffirm that the CTD alone can bind to the 30S and carry out ribosome anti-association. Therefore, we show that the CTD is the ribosome binding and anti-association domain, while the NTD is the major proofreading domain. Unpublished cryoEM structures from Prof. Ramakrishnan’s lab indicate that the NTD of IF3 pushes the i-tRNA at its elbow and helps in P site accommodation of the i-tRNA. We propose that when the codon-anticodon interaction is non-cognate or if the 3GC base pairs of the anticodon stem are not intact, then the dynamic action of the NTD destabilises the tRNA at the P site and leads to its rejection.
Part II: Proofreading function of mitochondrial IF3 (IF3mt)
Of the 13 protein-coding genes in mammalian mitochondria, 3 utilise the non-canonical AUA codon and one utilises the non-canonical start codon AUU. Since IF3mt does not possess many of the generally conserved residues implicated in proofreading, we decided to characterise the proofreading function of IF3mt and its role in initiation with non-canonical start codons. Structurally, IF3mt is similar to EcoIF3 with its N and C terminal domains joined by a linker region. However, IF3mt additionally possesses N- and C-terminal extensions which are generally disordered in structure. In vivo studies of mitochondrial translation factors have been mired by the lack of methodologies to manipulate mitochondria. We have developed an E. coli strain to study the proofreading functions of mitochondrial IF3 (IF3mt) with the help of reporter genes. Consistent with its function in mitochondria, IF3mt allowed promiscuous initiation from non-AUG codons. However, IF3mt avoided initiation with i-tRNAs lacking evolutionarily conserved 3GC pairs in anticodon stems. Interestingly, expression of IF3mt N-terminal domain or IF3mt devoid of its typical N-, and C-terminal extensions significantly improved its proofreading activity. Our immunoblot assays from polysome profile fractions indicate that the IF3mt derivative lacking extensions is capable of superior 30S ribosome binding. The two derivatives of IF3mt missing the Next (IF3mtΔNext) or both the Next and Cext (IF3mtΔNextCext) display an affinity for the 50S ribosome. We propose that the extensions of IF3mt may have evolved to reduce the affinity of IF3mt to the ribosome and thereby permit initiation with non-canonical start codons like AUU and AUA. Our studies suggest that E. coli provides an excellent heterologous model to study distinctive features of mitochondrial factors.
Part III: Fidelity of translation initiation in mycoplasma
One of the many singular features of mycoplasma is the presence of many anticodon stem variants of the i-tRNA across different species. In general, i-tRNAs are characterized by the presence of the typical feature of the conserved 3 consecutive GC base pairs (GC/GC/GC) in the anticodon stem. However, many mycoplasmal species have i-tRNAs with AU/GC/GC, GC/GC/GU or AU/GC/GU sequences. Interestingly, the mycoplasmal species which harbour the AU/GC/GU i-tRNA are also human pathogens. Therefore, we decided to investigate whether these organisms possess any unique features to accommodate the i-tRNA variants, by investigating the usage of Shine Dalgarno sequences and by carrying out multiple sequence alignments of genes encoding initiation factors, ribosomal proteins S9 and S13 and 16S rRNA. Since IF3 plays a crucial role in i-tRNA selection, we carried out computational analysis of mycoplasmal IF3 sequences, which revealed many interesting features. Most striking amongst them was the variation of the highly conserved R at position 131 in some species. Interestingly, these were the very mycoplasmal species which possessed the anticodon stem variant AU/GC/GU, suggesting a strong correlation between these two features. It is known that the R131P mutation of EcoIF3 is characterised by an enormous loss of proofreading activity. It seemed unusual that such compromised proofreading would be tolerated in the cell, so we decided to investigate other components of the translational machinery as well. The C-terminal SKR tail of the ribosomal protein S9, which contacts the P-site tRNA, is highly conserved across bacteria. Analysis of the C-terminal sequences of S9 proteins in various mycoplasmal species revealed a surprising variation- the presence of a TKR tail in strains with the AU/GC/GU tRNA. In this study we have investigated the co-occurrence of S9 and IF3 variations in i-tRNA selection in E. coli. We see that the R131P polymorphism of IF3 leads to a tremendous loss of proofreading, but this loss is significantly tempered by the presence of the S9 TKR variation. Our bioinformatics studies revealed that the mycoplasmal species which are sustained on AU/GC/GU i-tRNAs also tend to use a higher percentage of non-AUG codons. By means of our reporter assays in E. coli, we have shown once again that the R131P polymorphism of IF3 leads to a tremendous increase in initiation with the non-canonical start codon AUA, but this increase is significantly tempered by the presence of the S9 TKR variation
Role of conserved features of initiator tRNA and ribosome heterogeneity in translation initiation in Escherichia coli
No full textTranslation is one of the fundamental and core cellular processes catalysed by a ribonucleoprotein complex called ribosome. The process involves four major steps: initiation, elongation, termination and recycling. Initiation is the rate limiting step in translation, which determines the correct reading frame in an mRNA. Initiation occurs by formation of an initiation complex comprising 30S ribosomal subunit, mRNA, initiator tRNA, and initiation factors. The recruitment of 30S ribosomal subunit to the mRNA is aided by interaction between conserved RNA sequence called anti-Shine Dalgarno (aSD) in 16S rRNA and the Shine Dalgarno (SD) sequence in an mRNA present upstream of the start codon. Initiator tRNA (i-tRNA) is recruited directly to the ribosomal P-site with the help of initiation factor 2 (IF2). On the other hand all elongator tRNAs are brought to the ribosomal A-site by elongation factor Tu (EF-Tu). The P-site binding of i-tRNA has been attributed to two of its unique features. First, the CxA mismatch (in Escherichia coli) at 1x72 position, which is a major determinant for formylation of amino acid attached to i-tRNA. Formylation increases the affinity of i-tRNA to IF2 and prevents its binding to EF-Tu. Second, the presence of 3 consecutive GC base pairs (3GC pairs) in the anticodon stem of i-tRNA which is conserved in all the three domains of life. The i-tRNA lacking this feature is incompetent in initiation. However, the exact mechanism of how these two conserved features play a role in the fidelity of translation initiation is still not fully understood.
The work described in the thesis attempts to uncover the finer details of the fidelity at the step of initiation of protein synthesis using molecular genetics and biochemical tool
Studies on Nudix hydrolase proteins and crosstalk between DNA repair pathways
No full textMycobacterium tuberculosis, the causative agent of tuberculosis, has become a global health concern. This calls for a dire need to understand various aspects of mycobacterial physiology in order to design better strategies to control the infection. Inside the host macrophages, pathogen encounters high oxidative and nitrosative stress and their GC rich genomes, render them susceptible to exceptionally mutagenic base modifications like oxidation of guanine to 8-O-guanine and deamination of cytosine to uracil. To safeguard its DNA, the pathogen has evolved specialized mechanisms of DNA repair. MutT hydrolyzes 8-O-dGTP present in the nucleotide pool to its monophosphate form and eliminates chances of its misincorporation in the DNA. Even though 4 orthologs of MutT have been identified, the identity of a canonical MutT remains indeterminate in mycobacteria. The MutT proteins belong to Nudix hydrolase family of proteins. To further our understanding of MutT mediated 8-O-dGTP sanitization mechanisms in mycobacteria, we carried out biochemical and functional analysis of one of the mycobacterial Nudix hydrolase family proteins in the first part of the study. In the second part, we tested the functions of Nucleoside diphosphate kinase (NDK), known to maintain nucleotide pools, towards 8-O-dGTP using E. coli model system. In addition, Base Excision Repair (BER) and Nucleotide Excision Repair (NER) pathways are believed to play major roles in DNA repair in mycobacteria because of the absence of mismatch repair system and little contributions from RecA in eliciting the DNA damage response. In other organisms, NER has been observed to contribute in the repair of single nucleotide damage, facilitated by BER pathway specific proteins. In part III of the study, we have worked on a hypothesis that DNA damage repair by a uracil DNA glycosylase (UdgB) in mycobacteria invites NER pathway proteins to complete the repair
- …
