Anticodon sequence mutants of Escherichia coli initiator tRNA: effects of overproduction of aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase, and initiation factor 2 on activity in initiation.
Anticodon sequence mutants of Escherichia coli initiator tRNA initiate protein synthesis with codons other than AUG and amino acids other than methionine. Because the anticodon sequence is, in many cases, important for recognition of tRNAs by aminoacyl-tRNA synthetases, the mutant tRNAs are aminoacylated in vivo with different amino acids. The activity of a mutant tRNA in initiation in vivo depends on (i) the level of expression of the tRNA, (ii) the extent of aminoacylation of the tRNA, (iii) the extent of formylation of the aminoacyl-tRNA to formylaminoacyl-tRNA (fAA-tRNA), and (iv) the affinity of the fAA-tRNA for the initiation factor IF2 and the ribosome. Previously, using E. coli overproducing aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase, or IF2, we identified the steps limiting the activity in initiation of mutant tRNAs aminoacylated with glutamine and valine. Here, we have identified the steps limiting the activity of mutant tRNAs aminoacylated with isoleucine and phenylalanine. The combined results of experiments involving a variety of initiation codons (AUG, UAG, CAG, GUC, AUC, and UUC) provide support to the hypothesis that the ribosome.fAA-tRNA complex can act as an intermediate in initiation of protein synthesis. Comparison of binding affinities of various fAA-tRNAs (fMet-, fGln-, fVal-, fIle-, and fPhe-tRNAs) to IF2 using surface plasmon resonance supports the idea that IF2 can act as a carrier of fAA-tRNA to the ribosome. Other results suggest that the C1xA72 base pair mismatch, unique to eubacterial and organellar initiator tRNAs, may also be important for the binding of fAA-tRNA to IF2.
Mayer
Christine
C
Department of Biology, 68-671 Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Köhrer
Caroline
C
Kenny
Eimear
E
Prusko
Carsten
C
RajBhandary
Uttam L
UL
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D000926:Anticodon / Q000235:genetics*; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003062:Codon; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics*; D017930:Genes, Reporter; D019877:Hydroxymethyl and Formyl Transferases / Q000235:genetics*; D007700:Kinetics; D009154:Mutation; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D012343:RNA, Transfer / Q000235:genetics; D012358:RNA, Transfer, Met / Q000737:chemistry / Q000235:genetics*
D000926:Anticodon; D003062:Codon; D015852:Eukaryotic Initiation Factor-2; D012329:RNA, Bacterial; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D012343:RNA, Transfer; D019877:Hydroxymethyl and Formyl Transferases; C021561:methionyl-tRNA formyltransferase; D015500:Chloramphenicol O-Acetyltransferase; D000604:Amino Acyl-tRNA Synthetases