Effect of point mutations in the decoding site (C1400) region of 16S ribosomal RNA on the ability of ribosomes to carry out individual steps of protein synthesis.
In order to probe the relationship between structure and function of the ribosome, an in vitro system [Denman et al. (1988) Biochemistry (preceding paper in this issue)] was used to make a series of base changes around C1400, a residue known to be at the decoding site. Replacement of C1400 by U, A, or G, deletion of single bases at and to either side of C1400, and insertion of C or U next to C1400 were done. In a separate study, a mutant with seven extra nucleotides at the 3' end was constructed. The activity of these 11 mutants in A and P site binding and in initiation-dependent and initiation-independent peptide synthesis was analyzed. None of the base substitutions of C1400 were markedly inhibitory despite the almost complete conservation of this residue in ribosomal RNAs from a wide range of species. The insertions and deletions completely blocked initiation-dependent peptide synthesis but markedly stimulated the initiation-independent reaction. The effects on tRNA binding were variable. The extra stem and loop at the 3' end blocked initiation-dependent peptide synthesis but did not influence the other assays. The only modification to block all ribosomal function was the deletion of G1401. It appears that while the conserved and cross-linkable C1400 is not essential for function, the adjacent conserved G1401 is.
Denman
R
R
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
Nègre
D
D
Cunningham
P R
PR
Nurse
K
K
Colgan
J
J
Weitzmann
C
C
Ofengand
J
J
D002872:Chromosome Deletion; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D007700:Kinetics; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D012335:RNA, Ribosomal / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000096:biosynthesis* / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism*
D004251:DNA Transposable Elements; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins; D010444:Peptide Elongation Factor Tu