Mutants of Escherichia coli formylmethionine tRNA: a single base change enables initiator tRNA to act as an elongator in vitro.
We show that the absence of a Watson-Crick base pair at the end of the amino acid acceptor stem, which is a hallmark of all prokaryotic initiator tRNAs, is one of the key features that prevents them from acting as an elongator in protein synthesis. We generated mutants of Escherichia coli formylmethionine tRNA that have a base pair at the end of the acceptor stem. The mutants generated were C1----T1, which had a U.A base pair, A72----G72, which had a C.G base pair, and the C1A72----T1G72 double mutant, which lacked the base pair. After aminoacylation, the activity of these and other mutant initiator methionyl-tRNAs (Met-tRNAs) in elongation were assayed in a MS2 RNA-directed E. coli protein-synthesizing system and in binding to the elongation factor Tu (EF-Tu). Unlike wild-type initiator tRNA or the T1G72 double mutant, the T1 and G72 mutant Met-tRNAs were active in elongation, the G72 mutant being more active than the T1 mutant. The T1 and G72 mutant Met-tRNAs also formed a ternary complex with elongation factor EF-Tu.GTP, and their relative affinities for EF-Tu.GTP paralleled their activities in elongation. Combination of the T1 or G72 mutation with mutations in the GGG.CCC sequence conserved in the anticodon stem of initiator tRNAs led to a further increase in the activities of these mutant tRNAs in elongation such that one of these mutants was now almost as good an elongator as E. coli elongator methionine tRNA.
Seong
B L
BL
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
RajBhandary
U L
UL
D001483:Base Sequence; D003062:Codon; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics*; D006160:Guanosine Triphosphate / Q000378:metabolism; D066298:In Vitro Techniques; D010441:Peptide Chain Elongation, Translational; D010442:Peptide Chain Initiation, Translational; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics*; D012358:RNA, Transfer, Met / Q000235:genetics*; D013329:Structure-Activity Relationship
D003062:Codon; D010446:Peptide Fragments; D012345:RNA, Transfer, Amino Acid-Specific; D012358:RNA, Transfer, Met; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu