Roles of the ribosomal protein uS12 and the initiation factor 3 in the maintenance of fidelity of translation in Escherichia coli
The 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.