Translation of the genetic code requires attachment of tRNAs to their cognate amino acids. Errors during amino‐acid activation and tRNA esterification are corrected by aminoacyl‐tRNA ...synthetase‐catalyzed editing reactions, as extensively described for aliphatic amino acids. The contribution of editing to aromatic amino‐acid discrimination is less well understood. We show that phenylalanyl‐tRNA synthetase misactivates tyrosine and that it subsequently corrects such errors through hydrolysis of tyrosyl‐adenylate and Tyr‐tRNAPhe. Structural modeling combined with an in vivo genetic screen identified the editing site in the B3/B4 domain of the β subunit, 40 Å from the active site in the α subunit. Replacements of residues within the editing site had no effect on Phe‐tRNAPhe synthesis, but abolished hydrolysis of Tyr‐tRNAPhein vitro. Expression of the corresponding mutants in Escherichia coli significantly slowed growth, and changed the activity of a recoded β‐galactosidase variant by misincorporating tyrosine in place of phenylalanine. This loss in aromatic amino‐acid discrimination in vivo revealed that editing by phenylalanyl‐tRNA synthetase is essential for faithful translation of the genetic code.
Translation of the genetic code requires attachment of tRNAs to their cognate amino acids. Errors during amino-acid activation and tRNA esterification are corrected by aminoacyl-tRNA ...synthetase-catalyzed editing reactions, as extensively described for aliphatic amino acids. The contribution of editing to aromatic amino-acid discrimination is less well understood. We show that phenylalanyl-tRNA synthetase misactivates tyrosine and that it subsequently corrects such errors through hydrolysis of tyrosyl-adenylate and Tyr-tRNA(Phe). Structural modeling combined with an in vivo genetic screen identified the editing site in the B3/B4 domain of the beta subunit, 40 angstroms from the active site in the alpha subunit. Replacements of residues within the editing site had no effect on Phe-tRNA(Phe) synthesis, but abolished hydrolysis of Tyr-tRNA(Phe) in vitro. Expression of the corresponding mutants in Escherichia coli significantly slowed growth, and changed the activity of a recoded beta-galactosidase variant by misincorporating tyrosine in place of phenylalanine. This loss in aromatic amino-acid discrimination in vivo revealed that editing by phenylalanyl-tRNA synthetase is essential for faithful translation of the genetic code.
The prevalence and characteristics of small regulatory RNAs (sRNAs) have not been well characterized for Bacillus subtilis, an important model system for Gram-positive bacteria. However, B. subtilis ...was recently found to synthesize many candidate sRNAs during stationary phase. In the current study, we performed deep sequencing on Hfq-associated RNAs and found that a small subset of sRNAs associates with Hfq, an enigmatic RNA-binding protein that stabilizes sRNAs in Gram-negatives, but whose role is largely unknown in Gram-positive bacteria. We also found that Hfq associated with antisense RNAs, antitoxin transcripts, and many mRNA leaders. Several new candidate sRNAs and mRNA leader regions were also discovered by this analysis. Additionally, mRNA fragments overlapping with start or stop codons associated with Hfq, while, in contrast, relatively few full-length mRNAs were recovered. Deletion of hfq reduced the intracellular abundance of several representative sRNAs, suggesting that B. subtilis Hfq-sRNA interactions may be functionally significant in vivo. In general, we anticipate this catalog of Hfq-associated RNAs to serve as a resource in the functional characterization of Hfq in B. subtilis.
PDF
