Sequence analysis of the iclR gene encoding the repressor of the acetate operon in Salmonella typhimurium.
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Acetates↗
Biomedical subjects
D Nègre
Publications and source records attributed to D Nègre.
Relationship between guanosine tetraphosphate and accuracy of translation in Salmonella typhimurium.
In bacteria a high level of mistranslation is observed in amino acid starved rel-, but not rel+, strains, and mistranslation can be studied qualitatively by means of "stuttering" experiments in two-dimensional protein gels. It has been suggested that the low level of mistranslation that occurs in rel+ strains is assured by guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a nucleotide whose intracellular concentration greatly increases in rel+ cells under amino acid starvation. In the present study the relationship between level of ppGpp and mistranslation was analyzed by performing stuttering experiments in amino acid starved bacteria that contained either high or low levels of ppGpp. Three strains of Salmonella typhimurium were used in these experiments: a relA+ hisT+ strain (TA997), a relA+ hisT strain (TA1001), and a relA hisT strain (PD2). These strains were first characterized with respect to macromolecular syntheses and ppGpp levels under exponential growth and under amino acid starvation. Both rel+ strains exhibited stringent control over RNA synthesis. ppGpp accumulated to high levels when TA997 was starved for either of three amino acids. Starvation of TA1001 for histidine did not cause accumulation of ppGpp, whereas starvation for lysine and arginine produced high levels of ppGpp. Extracts from the three strains, obtained either under exponential growth or under amino acid starvation, were then subjected to two-dimensional electrophoretic anaylsis: mistranslation was observed whenever ppGpp was absent. In particular, starvation of TA1001 for histidine resulted in high mistranslation frequencies, while under lysine and arginine starvation mistranslation was undetectable, regardless of whether the cells were rel+ or rel-.(ABSTRACT TRUNCATED AT 250 WORDS)
Bacterial Proteins↗
In vitro assembly of 30S and 70S bacterial ribosomes from 16S RNA containing single base substitutions, insertions, and deletions around the decoding site (C1400).
An in vitro system developed for the site-specific mutagenesis of 16S RNA of Escherichia coli ribosomes [Krzyzosiak et al. (1987) Biochemistry 26, 2353-2364] was used to make 10 single base changes around C1400, a residue known to be at the decoding site. C1400 was replaced by U, A, or G, five single base deletions at and to either side of C1400 were made, and C or U was inserted next to C1400. Another mutant possessed seven additional nucleotides at the 3' end of the 16S RNA such that a stem and loop involving the anti-Shine-Dalgarno sequence could form. Each of the mutant RNAs was reconstituted with a complete mixture of 30S proteins to yield 30S ribosomes. Modified in vitro reconstitution conditions were required to obtain assembly of all of the synthetic ribosomes. Quantitative HPLC analysis of the protein content of each mutant showed that all of the proteins were present. The ability of synthetic 30S to form 70S particles under functional assay conditions was about 75% that of natural 30S and was unchanged by any of the mutations except for the deletion of G1401, which decreased the association activity under the standard conditions to 35-40% of synthetic 30S. That part of the ribosomal P site which interacts with the anticodon loop of tRNA was investigated by near-UV (greater than 300 nm) induced cross-linking of AcVal-tRNA. Cross-linking depended on both 30S subunits and the correct codon. The cross-linking yield of all mutants with a pyrimidine at position 1400 was equal to control isolated 30S, and the first-order rate constants for cross-linking of those mutants tested were like reconstituted natural 30S. The site of cross-linking for mutants with a C or U insertion between C1400 and G1401 was shifted to the inserted residue. Cross-linking to the base 5' to G1401 rather than to the residue 3' to C1399 indicates that G1401 is an important structural determinant of the P site.
Centrifugation, Density Gradient↗
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.
Chromosome Deletion↗
In vitro methylation of Escherichia coli 16S ribosomal RNA and 30S ribosomes.
Treatment of synthetic 30S particles lacking all of the normally methylated nucleotides with S-adenosyl-[3H]methionine and either an S100 or ribosomal high salt wash extract resulted in ribosome-dependent incorporation of [3H]methyl groups into trichloroacetic acid-insoluble material. No incorporation was observed when naturally methylated isolated 30S particles were used, showing that methylation at unnatural sites did not occur. Enzymatic hydrolysis of the labeled RNA to nucleosides followed by HPLC analysis identified the [3H]methylated residues. Activities for the formation of N6-methyladenosine, N6-dimethyladenosine, 5-methylcytidine (m5C), 3-methyluridine, and N2-methylguanosine were found. Fractionation by ammonium sulfate partially resolved the different activities. All of the fractions with m5C activity were 6-8 times more active on synthetic unmethylated 16S RNA than on synthetic 30S ribosomes, whereas the N2-methylguanosine activity preferred 30S ribosomes to 16S RNA by a factor of more than 10. The N6-methyladenosine and N6-dimethyladenosine activities were 30S ribosome-specific. The m5C activity present in the 55-85% ammonium sulfate fraction of the high salt wash yielded a maximum of 1.0 mol of m5C per mol of 16S RNA, although two m5C residues, positions 967 and 1407, are found in vivo. RNase protection by hybridization with the appropriate oligodeoxynucleotide identified the methylated residue as C-967. Methylation of m5C-967 did not require prior methylation of G-966, and methylation of A-1518 and A-1519 was not dependent on prior methylation of G-1516.
Base Sequence↗
Differential pattern of misreading induced by streptomycin in vitro.
A simplified, plasmid-directed coupled system was used to study the effect of streptomycin on the accuracy of natural messenger translation in vitro. The interaction of six different codons with their cognate tRNAs and 18 non-cognate tRNAs was analysed in the presence and absence of the antibiotic. Streptomycin appeared to modify, to a varying extent, the frequency of errors in codon-anticodon recognition. From this observation, some rules for mistranslation were inferred.
Cell-Free System↗
Promoter vectors with restriction-site banks.
New vectors harboring the promoter for the chloramphenicol acetyl transferase gene (cat promoter) have been constructed. These vectors are all derived from pJRD184 [Heusterspreute et al., Gene 39 (1985) 299-304], which contains a restriction-site bank. The cat promoter has been inserted at various positions and in reverse orientations so that almost all the restriction sites originally present on JRD184 can be used in cloning experiments. The expression of the aceK gene of Escherichia coli cloned under the control of the cat promoter has been tested. A large increase in the synthesis of the isocitrate dehydrogenase kinase, the aceK gene product, has demonstrated the efficiency of the newly constructed vectors.
Acetyltransferases↗
Accuracy of natural messenger translation: analysis of codon-anticodon recognition in a simplified cell-free system.
A simplified plasmid-directed coupled system [Robakis, N., Cenatiempo, Y., Meza-Basso, L., Brot, N., & Weissbach, H. (1983) Methods Enzymol. 101, 690-706] was used to study the accuracy of natural messenger translation in vitro. In this system, protein synthesis is limited to the formation of the N-terminal di- or tripeptide of the gene product. Such a control is obtained by restricting the supply of aminoacyl-tRNAs in the assay medium to those corresponding specifically to the first two or three triplets in the mRNA coding sequence. We analyzed comparatively the interaction of 6 different codons with their cognate tRNAs and 18 noncognate tRNAs able to recognize triplets differing from the legitimate sequences by one base only. Special attention was paid to the single base errors occurring at the first and second codon positions during ribosomal selection of aminoacyl-tRNA molecules. The noncognate tRNAs were assayed either in the absence of the legitimate tRNAs or under competition conditions. They were chosen so that all the possibilities for misreading any particular base as each of the other three bases could be studied. First, it was mainly observed that translation mistakes can be equally detected in the first and second codon positions; there is no compelling evidence for a most or least accurate site. Second, pyrimidines seem to be read more accurately than purines. In particular, U cannot be read as either C or G, and C can hardly be mistaken for any other base.(ABSTRACT TRUNCATED AT 250 WORDS)
Anticodon↗