title: Highly conserved base A55 of 16S ribosomal RNA is important for the elongation cycle of protein synthesis.
abstract: Accurate decoding of mRNA requires the precise interaction of protein factors and tRNAs with the ribosome. X-ray crystallography and cryo-electron microscopy have provided detailed structural information about the 70S ribosome with protein factors and tRNAs trapped during translation. Crystal structures showed that one of the universally conserved 16S rRNA bases, A55, in the shoulder domain of the 30S subunit interacts with elongation factors Tu and G (EF-Tu and EF-G, respectively). The exact functional role of A55 in protein synthesis is not clear. We changed A55 to U and analyzed the effect of the mutation on the elongation cycle of protein synthesis using functional assays. Expression of 16S rRNA with the A55U mutation in cells confers a dominant lethal phenotype. Additionally, ribosomes with the A55U mutation in 16S rRNA show substantially reduced in vitro protein synthesis activity. Equilibrium binding studies showed that the A55U mutation considerably inhibited the binding of the EF-Tu·GTP·tRNA ternary complex to the ribosome. Furthermore, the A55U mutation slightly inhibited the peptidyl transferase reaction, the binding of EF-G·GTP to the ribosome, and mRNA-tRNA translocation. These results indicate that A55 is important for fine-tuning the activity of the ribosome during the elongation cycle of protein synthesis.
authors.affiliation: Department of Chemistry and Biochemistry, University of California at San Diego , 9500 Gilman Drive, La Jolla, California 92093-0314, United States.
authors.forename: Bhubanananda
authors.initials: B
authors.lastname: Sahu
authors.forename: Prashant K
authors.initials: PK
authors.lastname: Khade
authors.forename: Simpson
authors.initials: S
authors.lastname: Joseph
mesh_terms: D000249:Adenosine Monophosphate / Q000235:genetics; D020653:Peptide Elongation Factor G / Q000378:metabolism; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010458:Peptidyl Transferases / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics*; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D014542:Uridine Monophosphate / Q000235:genetics
chemical_list: D020653:Peptide Elongation Factor G; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S; D000249:Adenosine Monophosphate; D012343:RNA, Transfer; D014542:Uridine Monophosphate; D010458:Peptidyl Transferases; D010444:Peptide Elongation Factor Tu
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