Accurate translation of the genetic code is critical to ensure expression of proteins with correct amino acid sequences. Certain tRNAs can cause a shift out of frame (i.e., frameshifting) due to imbalances in tRNA concentrations, lack of tRNA modifications or insertions or deletions in tRNAs (called frameshift suppressors). Here, we determined the structural basis for how frameshift-suppressor tRNASufA6 (a derivative of tRNAPro) reprograms the mRNA frame to translate a 4-nt codon when bound to the bacterial ribosome. After decoding at the aminoacyl (A) site, the crystal structure of the anticodon stem-loop of tRNASufA6 bound in the peptidyl (P) site reveals ASL conformational changes that allow for recoding into the +1 mRNA frame. Furthermore, a crystal structure of full-length tRNASufA6 programmed in the P site shows extensive conformational rearrangements of the 30S head and body domains similar to what is observed in a translocation intermediate state containing elongation factor G (EF-G). The 30S movement positions tRNASufA6 toward the 30S exit (E) site disrupting key 16S rRNA–mRNA interactions that typically define the mRNA frame. In summary, this tRNA-induced 30S domain change in the absence of EF-G causes the ribosome to lose its grip on the mRNA and uncouples the canonical forward movement of the tRNAs during elongation.

Background: Adenoviruses use the short non-coding transcript VA RNA I to inhibit host antiviral kinase PKR. Results: VA RNA I contains a pH-and Mg 2ϩ -sensitive tertiary structure that, unexpectedly, is not required for PKR inhibition. Conclusion: Structural requirements for an RNA inhibitor of PKR are simpler than appreciated previously. Significance: These findings explain how non-coding RNAs of varied sequence and structure can efficiently inhibit PKR.

RNA methyltransferases (MTases) are important players in the biogenesis and regulation of the ribosome, the cellular machine for protein synthesis. RsmC is a MTase that catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to G1207 of 16S rRNA. Mutations of G1207 have dominant lethal phenotypes in Escherichia coli, underscoring the significance of this modified nucleotide for ribosome function. Here we report the crystal structure of E. coli RsmC refined to 2.1 Å resolution, which reveals two homologous domains tandemly duplicated within a single polypeptide. We characterized the function of the individual domains and identified key residues involved in binding of rRNA and SAM, and in catalysis. We also discovered that one of the domains is important for the folding of the other. Domain duplication and subfunctionalization by complementary degeneration of redundant functions (in particular substrate binding versus catalysis) has been reported for many enzymes, including those involved in RNA metabolism. Thus, RsmC can be regarded as a model system for functional streamlining of domains accompanied by the development of dependencies concerning folding and stability.

Modifications in the tRNA anticodon loop, adjacent to the three-nucleotide anticodon, influence translation fidelity by stabilizing the tRNA to allow for accurate reading of the mRNA genetic code. One example is the N1-methylguaonosine modification at guanine nucleotide 37 (m1G37) located in the anticodon loop, immediately adjacent to the anticodon nucleotides 34-36. The absence of m1G37 in tRNAPro causes +1 frameshifting on polynucleotide, slippery codons. Here, we report structures of the bacterial ribosome containing tRNAPro bound to either cognate or slippery codons to determine how the m1G37 modification prevents mRNA frameshifting. The structures reveal that certain codon-anticodon contexts and m1G37 destabilize interactions of tRNAPro with the peptidyl site, causing large conformational changes typically only seen during EF-G mediated translocation of the mRNA-tRNA pairs. These studies provide molecular insights into how m1G37 stabilizes the interactions of tRNAPro with the ribosome and the influence of slippery codons on the mRNA reading frame.

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Highlights d Autotaxin (Atx) is a key regulator of satellite cell function d Conditional deletion of Atx in satellite cells impairs skeletal muscle regeneration d Atx-LPA induces S6K-mTOR signaling via LPAR1 d Atx-LPA promotes muscle hypertrophy

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bMethylation of bacterial 16S rRNA within the ribosomal decoding center confers exceptionally high resistance to aminoglycoside antibiotics. This resistance mechanism is exploited by aminoglycoside producers for self-protection while functionally equivalent methyltransferases have been acquired by human and animal pathogenic bacteria. Here, we report structural and functional analyses of the Sorangium cellulosum So ce56 aminoglycoside resistance-conferring methyltransferase Kmr. Our results demonstrate that Kmr is a 16S rRNA methyltransferase acting at residue A1408 to confer a canonical aminoglycoside resistance spectrum in Escherichia coli. Kmr possesses a class I methyltransferase core fold but with dramatic differences in the regions which augment this structure to confer substrate specificity in functionally related enzymes. Most strikingly, the region linking core ␤-strands 6 and 7, which forms part of the S-adenosyl-L-methionine (SAM) binding pocket and contributes to base flipping by the m 1 A1408 methyltransferase NpmA, is disordered in Kmr, correlating with an exceptionally weak affinity for SAM. Kmr is unexpectedly insensitive to substitutions of residues critical for activity of other 16S rRNA (A1408) methyltransferases and also to the effects of by-product inhibition by S-adenosylhomocysteine (SAH). Collectively, our results indicate that adoption of a catalytically competent Kmr conformation and binding of the obligatory cosubstrate SAM must be induced by interaction with the 30S subunit substrate. Gene pools in soil and aquatic microbial communities represent largely unexplored reservoirs of antibiotic resistance (1, 2). An analysis of the resistance mechanisms present in these communities may reveal new insights into the origins, activities, and potential for mobilization of antibiotic resistance determinants to pathogenic bacterial populations. In particular, myxobacteria of the genus Sorangium have become a major source of secondary metabolites over the last several decades alongside the actinobacteria and fungi (3, 4). The model strain Sorangium cellulosum So ce56 possesses a 13.1-Mb genome with 17 gene clusters for secondary metabolites (5). Associated with this tremendous biosynthetic power is a high resistance potential against multiple antibiotics. For example, all Sorangium strains can grow in the presence of exceptionally high concentrations of kanamycin through the action of a single, specific aminoglycoside resistance-conferring 16S rRNA methyltransferase, Kmr (6).In common with most aminoglycosides, kanamycin binds within helix (h) 44 of the 16S rRNA in the bacterial small (30S) ribosomal subunit and induces errors in mRNA decoding (7-9). Aminoglycoside-producing bacteria typically protect themselves from self-intoxication by expression of aminoglycoside resistance-conferring methyltransferases that modify the drug binding site. Two subfamilies of 16S rRNA methyltransferases introduce distinct 16S rRNA modifications at N7 of guanosine 1405 (m 7 G1405) or N1 of adenosine 1408 (m 1 A140...