A fundamental requirement of a protein synthesis system is that it decode and translocate successive mRNA triplets with speed and accuracy. The recent high-resolution X-ray and cryo-EM structures of bacterial and archaeal ribosomes have provided enormous insights into how the decoding center of the small ribosomal subunit accurately discriminates between correct and incorrect tRNA substrates. Meanwhile, our genetic analyses of E. coli ribosomes have shown that in addition to the decoding center, accurate decoding and the ability to maintain reading frame is affected by the structure of regions of the ribosome distant from the site of codon-anticodon interaction. The function of the decoding region is thus influenced by many regions of the ribosome and the sequential steps of translation (decoding, peptide bond formation and translocation) are interconnected. The several bridges between the large and small ribosomal subunits are believed to be essential elements in the transmission of information between the ribosomal subunits and coordination of their activities. We have shown that changes in the 50S component of one of the largest bridges (B2a) alters the structure of the 30S subunit and perturbs the accuracy of translation and we are now carrying out a systematic mutagenesis of the regions of rRNA and r-proteins involved in inter-subunit bridge formation. Future work will use genetic biochemical and structural approaches to uncover the function of the inter-subunit bridges and gain insights into the coordination of ribosomal components and activities during the translation cycle.
A second interest is in the mechanism of tmRNA-mediated trans-translation. In bacteria, ribosome stalling on truncated mRNAs lacking stop codons recruits a small, stable RNA, tmRNA, to the ribosomal A site where it first acts as a tRNA. Subsequently, tmRNA assumes the role of a messenger RNA and translation switches from the truncated mRNA to tmRNA. This process recycles stalled ribosomes and tags incomplete polypeptides for degradation. Our particular interests are in the signals that recruit tmRNA to the ribosome, the interactions between tmRNA and the translation machinery and the mechanism of switching from tRNA to mRNA function.
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