Functional effects of a G to U base change at position 530 in a highly conserved loop of Escherichia coli 16S RNA.
Any base change at position 530 introduced into Escherichia coli on a multicopy plasmid leads to cell death [Powers & Noller (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1042-1046]. It was suggested that these mutants cannot carry out chain elongation. To define more precisely the function of base 530, we have studied ribosomes in which G530 was mutated to U530. In vivo, U530 16S rRNA was incorporated into 30S subunits and could combine with 50S to make 70S ribosomes. 16S rRNA in vitro transcripts containing U530 were assembled into 30S ribosomes, and their activity was tested in defined steps of protein synthesis. Mutant 30S ribosomes were as active as wild-type in poly(U)-dependent poly(Phe) synthesis, P- and A-site tRNA binding, and 30S initiation complex formation. 30S initiation complexes, in the presence of 50S, could react with puromycin like the wild-type. The rate, extent, and position of cross-linking of AcVal-tRNA in the P site to 16S RNA were identical in mutant and wild-type ribosomes. Although there appeared to be no defect in 70S initiation complex formation or in direct A-site binding of Phe-tRNA dependent on poly(U), U530 30S ribosomes were nevertheless defective in carrying out synthesis of fMet-Val dipeptide using natural mRNA. Mutant 30S ribosomes were also refractory to streptomycin-induced misreading although no misreading was observed in its absence.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biology, Haverford College, Pennsylvania 19041.
M
M
Santer
U
U
Santer
K
K
Nurse
A
A
Bakin
P
P
Cunningham
M
M
Zain
D
D
O'Connell
J
J
Ofengand
D001483:Base Sequence; D002876:Chromosomes, Bacterial; D004151:Dipeptides / Q000096:biosynthesis; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D006147:Guanine; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011072:Poly U / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics* / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D014498:Uracil
D004151:Dipeptides; D012336:RNA, Ribosomal, 16S; D012346:RNA, Transfer, Amino Acyl; D012360:RNA, Transfer, Phe; C016200:tRNA, N-acetylvaline-; D011072:Poly U; C042204:N-formylmethionylvaline; D014498:Uracil; D006147:Guanine