Global protein synthesis is emerging as an important player in the context of aging and age-related diseases. However, the intricate molecular networks that regulate protein synthesis are poorly understood. Here, we report that SIRT6, a nuclear-localized histone deacetylase represses global protein synthesis by transcriptionally regulating mTOR signalling via the transcription factor Sp1, independent of its deacetylase activity. Our results suggest that SIRT6 deficiency increases protein synthesis in mice. Further, multiple lines of in vitro evidence suggest that SIRT6 negatively regulates protein synthesis in a cell-autonomous fashion and independent of its catalytic activity. Mechanistically, SIRT6 binds to the zinc finger DNA binding domain of Sp1 and represses its activity. SIRT6 deficiency increased the occupancy of Sp1 at key mTOR signalling gene promoters resulting in enhanced expression of these genes and activation of the mTOR signalling pathway. Interestingly, inhibition of either mTOR or Sp1 abrogated the increased protein synthesis observed under SIRT6 deficient conditions. Moreover, pharmacological inhibition of mTOR restored cardiac function in muscle-specific SIRT6 knockout mice, which spontaneously develop cardiac hypertrophy. Overall, these findings have unravelled a new layer of regulation of global protein synthesis by SIRT6, which can be potentially targeted to combat aging-associated diseases like cardiac hypertrophy.

Eubacterial translation initiation involves assembly of tRNAfMet, mRNA, initiation factors (IFs) and 30S ribosome in a 30S pre-initiation complex (30S pre-IC), which rearranges and joins 50S ribosome to form 70S IC. Upon releasing IFs, 70S IC becomes elongation-competent 70S. The direct recruitment of initiator tRNA (tRNAfMet) into the ribosomal P-site, crucial in accurate initiation of translation, is attributed to two conserved features of tRNAfMet: (i) formylation of amino acid attached to it and, (ii) the presence of three consecutive G-C base pairs (3GC base pairs) in the anticodon stem. However, the precise roles of these two conserved features of tRNAfMet during the various steps of initiation remain unclear. Using natural and engineered tRNAs, we show that the 3GC pairs license tRNAfMet transitions from 30S to 70S IC and then to elongation-competent 70S by release of IF3. Of the 3GC pairs, the middle GC pair (G30-C40), or merely G30 (in a specific context) suffices in this role and is essential for the sustenance of Escherichia coli. Furthermore, rescue of formylase deficient E. coli by overproduced tRNAfMet reveals that the feature of formylation licenses initial targeting of tRNAfMet to 30S ribosome.

Initiator tRNAs (i-tRNAs) possess highly conserved three consecutive GC base pairs (GC/GC/GC, 3GC pairs) in their anticodon stems. Additionally, in bacteria and eukaryotic organelles, the amino acid attached to i-tRNA is formylated by Fmt to facilitate its targeting to 30S ribosomes. Mutations in GC/GC/GC to UA/CG/AU in i-tRNA CUA/ 3GC do not affect its formylation. However, the i-tRNA CUA/ 3GC is non-functional in initiation. Here, we characterised an Escherichia coli strain possessing an amber mutation in its fmt gene ( fmt am274 ), which affords initiation with i-tRNA CUA/ 3GC . Replacement of fmt with fmt am274 in the parent strain results in production of truncated Fmt, accumulation of unformylated i-tRNA, and a slow growth phenotype. Introduction of i-tRNA CUA/ 3GC into the fmt am274 strain restores accumulation of formylated i-tRNAs and rescues the growth defect of the strain. We show that i-tRNA CUA/ 3GC causes a low level suppression of am274 in fmt am274 . Low levels of cellular Fmt lead to compromised efficiency of formylation of i-tRNAs, which in turn results in distribution of the charged i-tRNAs between IF2 and EF-Tu allowing the plasmid borne i-tRNA CUA/ 3GC to function at both the initiation and elongation steps. We show that a speedy formylation of i-tRNA population is crucial for its preferential binding (and preventing other tRNAs) into the P-site.

Initiator tRNAs are special in their direct binding to the ribosomal P-site due to the hallmark occurrence of the three consecutive G-C base pairs (3GC pairs) in their anticodon stems. How the 3GC pairs function in this role, has remained unsolved. We show that mutations in either the mRNA or 16S rRNA leading to extended interaction between the Shine-Dalgarno (SD) and anti-SD sequences compensate for the vital need of the 3GC pairs in tRNA fMet for its function in Escherichia coli. In vivo, the 3GC mutant tRNA fMet occurred less abundantly in 70S ribosomes but normally on 30S subunits. However, the extended SD:anti-SD interaction increased its occurrence in 70S ribosomes. We propose that the 3GC pairs play a critical role in tRNA fMet retention in ribosome during the conformational changes that mark the transition of 30S preinitiation complex into elongation competent 70S complex. Furthermore, treating cells with kasugamycin, decreasing ribosome recycling factor (RRF) activity or increasing initiation factor 2 (IF2) levels enhanced initiation with the 3GC mutant tRNA fMet , suggesting that the 70S mode of initiation is less dependent on the 3GC pairs in tRNA fMet . 3GC base pairsnitiation of protein synthesis, assisted by initiation factors, is a highly regulated process in all life forms. In eubacteria, binding of both the initiator tRNA (tRNA fMet ) and mRNA to the small ribosomal subunit (30S) leads to the formation of a 30S preinitiation complex primarily with the help of the three initiation factors (IF1, IF2, and IF3). This stage is then followed by docking of the large ribosomal subunit (50S) to ultimately produce an elongation competent 70S complex upon the departure of all of the three initiation factors (1). The localization of mRNA onto the 30S subunit is facilitated by a purine rich sequence (Shine-Dalgarno, SD sequence), located upstream of the start codon, by its pairing with a complementary sequence (anti-SD sequence) at the 3′-terminus of the 16S rRNA (1, 2). The tRNA fMet binding to ribosome is aided by the unique features it possesses. A virtually universal feature of all of the initiator tRNAs, the presence of three consecutive G-C base pairs (G 29 G 30 G 31 :C 39 C 40 C 41 , referred to as 3GC pairs) in the anticodon stem is known to be important for their preferential binding in the ribosomal P-site (3, 4). Mutations in the 3GC pairs result in poor binding of tRNA fMet to the ribosomal P-site (3, 4).However, the mechanism of how the 3GC pairs help in binding of tRNA fMet into the ribosome has remained unclear. In the crystal structure of the initiator tRNA bound 70S ribosome, it was seen that the universally conserved A1339 and G1338 residues of 16S rRNA establish A-minor interactions with the first GC (G 29 -C 41 ) and middle GC (G 30 -C 40 ) pairs, respectively (5). Although these interactions were seen as suboptimal, the IF3 induced conformational changes may optimize these interactions (6). Another study showed that a major role of IF3 is to uniformly increase the rate of dissociation of...

Initiator tRNAs (i-tRNAs) are characterized by the presence of three consecutive GC base pairs (GC/GC/GC) in their anticodon stems in all domains of life. However, many mycoplasmas possess unconventional i-tRNAs wherein the highly conserved sequence of GC/GC/GC is represented by AU/GC/GC, GC/GC/GU or AU/GC/GU. These mycoplasmas also tend to preferentially utilize non-AUG initiation codons. To investigate if initiation with the unconventional i-tRNAs and non-AUG codons in mycoplasmas correlated with the changes in the other components of the translation machinery, we carried out multiple sequence alignments of genes encoding initiation factors (IF), 16S rRNAs, and the ribosomal proteins such as uS9, uS12 and uS13. In addition, the occurrence of Shine-Dalgarno sequences in mRNAs was analyzed. We observed that in the mycoplasmas harboring AU/GC/GU i-tRNAs, a highly conserved position of R131 in IF3, is represented by P, F or Y and, the conserved C-terminal tail (SKR) of uS9 is represented by the TKR sequence. Using the Escherichia coli model, we show that the change of R131 in IF3 optimizes initiation with the AU/GC/GU i-tRNAs. Also, the SKR to TKR change in uS9 was compatible with the R131P variation in IF3 for initiation with the AU/GC/GU i-tRNA variant. Interestingly, the mycoplasmas harboring AU/GC/GU i-tRNAs are also human pathogens. We propose that these mycoplasmas might have evolved a relaxed translational apparatus to adapt to the environment they encounter in the host.