Characterization of GE82832, a peptide inhibitor of translocation interacting with bacterial 30S ribosomal subunits

GE82832, a secondary metabolite produced by Streptosporangium cinnabarinum (strain GE82832), has been identified as a translational inhibitor by in vitro screening of a library of natural products. Secondary functional tests specific for individual steps of the translational pathway demonstrated that translocation is the specific target of GE82832. Chemical probing in situ demonstrated that this antibiotic protects bases A1324 and A1333 and exposes C1336 of 16S rRNA, thereby indicating that its binding site is located on the head of the 30S ribosomal subunit. The ribosomal location of GE82832, near ribosomal protein S13 and G1338, two elements of the small subunit that are part of or close to the B1a intrasubunit bridge, suggests that translocation inhibition results from an altered dynamics of 30S-50S ribosomal subunit interaction.     

Construction of an in vivo nonsense readthrough assay system and functional analysis of ribosomal proteins S12, S4, and S5 in Bacillus subtilis

To investigate the function of ribosomal proteins and translational factors in Bacillus subtilis, we developed an in vivo assay system to measure the level of nonsense readthrough by utilizing the LacZ-LacI system. Using the in vivo nonsense readthrough assay system which we developed, together with an in vitro poly(U)-directed cell-free translation assay system, we compared the processibility and translational accuracy of mutant ribosomes with those of the wild-type ribosome. Like Escherichia coli mutants, most S12 mutants exhibited lower frequencies of both UGA readthrough and missense error; the only exception was a mutant (in which Lys-56 was changed to Arg) which exhibited a threefold-higher frequency of readthrough than the wild-type strain. We also isolated several ribosomal ambiguity (ram) mutants from an S12 mutant. These ram mutants and the S12 mutant mentioned above (in which Lys-56 was changed to Arg) exhibited higher UGA readthrough levels. Thus, the mutation which altered Lys-56 to Arg resulted in a ram phenotype in B. subtilis. The efficacy of our in vivo nonsense readthrough assay system was demonstrated in our investigation of the function of ribosomal proteins and translational factors.     

Translational misreading: mutations in translation elongation factor 1alpha differentially affect programmed ribosomal frameshifting and drug sensitivity.

The translation elongation feactor 1alpha (EF-1alpha) catalyzes the critical step of delivering aminoacyl-tRNAs to the elongating ribosome. A series of Saccharomyces cerevisiae strains containing mutant alleles of the TEF2 gene encoding EF-1alpha have phenotypes consistent with effects on cellular processes related to translation. These include (1) conditional growth defects, (2) antibiotic sensitivity or resistance, (3) altered +1 or -1 ribosomal frameshifting efficiencies, and (4) altered maintenance of the killer phenotype. Although all the mutant alleles were isolated as dominant +1 frameshift suppressors, the effects of these mutations on the cell are quite different when present as the only form of EF-1alpha. Allele-specific effects are observed with regard to their ability to alter the efficiency of programmed +1 frameshifting as opposed to programmed -1 ribosomal frameshifting. The significantly altered efficiency of -1 frameshifting in strains containing the TEF2-4 and TEF2-9 mutant alleles further correlates with a reduced ability to maintain the killer phenotype and the M1 satellite virus of L-A, an in vivo assay of translational fidelity. In light of the proposed models regarding the different A- and P-site occupancy states required for +1 or -1 ribosomal frameshifting, these results aid analysis of interactions between EF-1alpha and the translational apparatus.     

Ribosomal suppressors and antisuppressors in Podospora anserina: Altered susceptibility to paromomycin and relationships between genetic and phenotypic suppression

Informational suppressors and antisuppressors have been previously isolated in Podospora anserina, and their properties suggest that they could be ribosomal mutants involved in the control of translational fidelity. In this paper we present results concerning relationships between these mutants and paromomycin, an aminoglycoside antibiotic known to stimulate translational errors. The mutants were found to manifest an altered growth sensitivity to this drug as compared with the wild-type strain: Most of the suppressors were more sensitive and, in contrast, most of the antisuppressors were more resistant to paromomycin. Moreover, phenotypic suppression of an auxotrophic mutation by paromomycin was observed only if a suppressor and an antisuppressor had been introduced in the strain. These results suggest that ambiguity levels could be altered in the suppressor and antisuppressor strains. In addition, paromomycin was shown to abolish sporulation, which suggests relationships between mistranslation and a step of cellular differentiation.This work was supported by a DGRST grant and by a NATO grant.     

Functional interactions in vivo between suppressor tRNA and mutationally altered ribosomal protein S4

Summary Ribosomal mutants (rpsD) which are associated with a generally increased translational ambiguity were investigated for their effects in vivo on individual tRNA species using suppressor tRNAs as models. It was found that nonsense suppression is either increased, unaffected or decreased depending on the codon context and the rpsD allele involved as well as the nature of the suppressor tRNA. Missense suppression of AGA and AGG by glyT(SuAGA/G) tRNA as well as UGG by glyT(SuUGG-8) tRNA is unaffected whereas suppression of UGG by glyT(SuUGA/G) or glyV(SuUGA/G) tRNA is decreased in the presence of an rpsD mutation. The effects on suppressor tRNA are thus not correlated with the ribosomal ambiguity (Ram) phenotype of the rpsD mutants used in this study. It is suggested that the mutationally altered ribosomes are changed in functional interactions with the suppressor tRNA itself rather than with the competing translational release factor(s) or cognate aminoacyl tRNA. The structure of suppressor tRNA, particularly the anticodon loop, and the suppressed codon as well as the codon context determine the allele specific functional interactions with these ribosomal mutations.     

Genetic mapping of a mutation causing an alteration in Bacillus subtilis ribosomal protein S4

Summary A mutation causing an alteration in Bacillus subtilis ribosomal protein S4 was mapped by transformation and PBS-1 transduction to a site between aroG and argA, a region of the B. subtilis chromosome not previously demonstrated to contain ribosomal protein genes. The S4 mutation conferred a spore-plus phenotype in a streptomycinresistant, spore-minus genetic background. The altered protein was detectable by polyacrylamide gel electrophoresis of ribosomal proteins of recombinants scored for the sporeplus phenotype in genetic crosses.     

A transcript map of an 800-kb region on human chromosome 11q13, part of the candidate region for SCA5 and BBS1

The exon-amplification method was used to identify putative transcribed sequences from an 800-kb region that includes the genes for phospholipase Cβ3 and PYGM on human chromosome 11q13. The clone contig consisted of ten cosmids, three bacterial artificial chromosomes, and one P1 artificial chromosome. A total of 83 exons were generated of which 23 were derived from known genes and expressed sequence tags (ESTs). Five different EST cDNA clones were identified and mapped on the contig. One is a homolog of the human p70S6 kinase (p70s6 k) gene whose function involves the translational regulation of ribosomal protein synthesis and thereby impacts on ribosomal biogenesis. The gene for p70s6 k is expressed universally, including within adipose cells and retina, and it could play a role in Bardet-Biedl syndrome type 1, which has been mapped to 11q13. Received: 22 July 1998 / Accepted: 24 August 1998     

Ribosomal protein limitations in Escherichia coli under conditions of high translational activity

Details of the mechanism for ribosome synthesis have been incorporated in the single-cell Escherichia coli model, which enable us to predict the amount of protein synthesizing machinery under different environmental conditions. The predictions agree quite well with available experimental data. The model predicts that ribosomal protein limitations are important when the translational apparatus is in high demand. Ribosomal RNA synthesis is induced by an increase in translational activity, which, in turn, stimulates ribosomal protein synthesis. However, as the demand increases still more, the ribosomal protein mRNA must compete with the plasmid mRNA for ribosomes, and the efficiency of translation of ribosomal proteins is reduced. (c) 1994 John Wiley & Sons, Inc.     

The isolation and characteristics of the biological and genetic properties of neamin-resistant mutants of Salmonella abortus-ovis that are promising for vaccine creation

Neamine-resistant mutants were obtained from S. abortus ovis virulent strain. These mutants were divided into three classes according to their sensitivity to streptomycin: mutants completely retaining their sensitivity, mutants sensitive to moderate and high doses of the antibiotic. On the basis of genetic analysis carried out with the use of bacteriophage P22, the Near mutation of class Near 100 Strr 500 mutants was identified as nea B, and the Near mutation of class Near 100 Strs, as nea A. The study showed a decreased virulence of Salmonella transductants that acquired both neamine-resistant mutation of the two classes and streptomycin-resistant mutation. The streptomycin-resistant mutation produced no changes in the virulence of these bacteria. According to the results of experiments on mice, mutants of the two classes under study were found to possess protective activity.     

Modulation of ribosomal recruitment to 5'-terminal start codons by translation initiation factors IF2 and IF3

Sequence determinants and structural features of the RNA govern mRNA-ribosome interaction in bacteria. However, ribosomal recruitment to leaderless mRNAs, which start directly with the AUG start codon and do not bear a Shine-Dalgarno sequence like canonical mRNAs, does not appear to rely on 16S rRNA-mRNA interactions. Here, we have studied the effects of translation initiation factors IF2 and IF3 on 30S initiation at a 5'-terminal AUG and at a competing downstream canonical ribosome binding site. We show that IF2 affects the forward kinetics of 30S initiation complex formation at the 5'-terminal AUG as well as the stability of these complexes. Moreover, the IF2:IF3 molar ratio was found to play a decisive role in translation initiation of a leaderless mRNA both in vitro and in vivo indicating that the translational efficiency of an mRNA is not only intrinsically determined but can be altered depending on the availability of components of the translational machinery.     

Genetic analysis of a streptomycin-resistant oligosporogenous Bacillus subtilis mutant

Strain SRB15T+, a streptomycin-resistant, oligosporogenous mutant of Bacillus subtilis, contains two mutations, fun and strR. These mutations were mapped by PBS-1 mediated transduction and by transformation to two different sites in the cysA-linked region of the B. subtilis chromosome. The fun mutation mapped very close to rpsLl, a classic strA mutation, whereas strR mapped to a site distal to rpsE. The effects of these mutations on growth, sporulation, and streptomycin resistance in vivo and in vitro were determined. The fun mutation gave a different phenotype than did the rpsLl mutation and caused altered migration of a ribosomal protein which was identified as S12, the protein encoded by rpsL. It therefore appears that fun is an allele of the rpsL gene.     

The human mitochondrial ribosomal protein genes: mapping of 54 genes to the chromosomes and implications for human disorders.

Mitochondria possess their own translational machinery, which is composed of components distinct from their cytoplasmic counterparts. To investigate the possible involvement of mitochondrial ribosomal defects in human disease, we mapped nuclear genes that encode mitochondrial ribosomal proteins (MRPs). We generated sequence-tagged sites (STSs) of individual MRP genes that were able to be detected by PCR. They were placed on an STS content map of the human genome by typing of radiation hybrid panels. We located 54 MRP genes on the STS-content map and assigned these genes to cytogenetic bands of the human chromosomes. Although mitochondria are thought to have originated from bacteria, in which the genes encoding ribosomal proteins are clustered into operons, the mapped MRP genes are widely dispersed throughout the genome, suggesting that transfer of each MRP gene to the nuclear genome occurred individually. We compared the assigned positions with candidate regions for mendelian disorders and found certain genes that might be involved in particular diseases. This map provides a basis for studying possible roles of MRP defects in mitochondrial disorders.     

Halting a cellular production line: responses to ribosomal pausing during translation.

Cellular protein synthesis is a complex polymerization process carried out by multiple ribosomes translating individual mRNAs. The process must be responsive to rapidly changing conditions in the cell that could cause ribosomal pausing and queuing. In some circumstances, pausing of a bacterial ribosome can trigger translational abandonment via the process of trans-translation, mediated by tmRNA (transfer-messenger RNA) and endonucleases. Together, these factors release the ribosome from the mRNA and target the incomplete polypeptide for destruction. In eukaryotes, ribosomal pausing can initiate an analogous process carried out by the Dom34p and Hbs1p proteins, which trigger endonucleolytic attack of the mRNA, a process termed mRNA no-go decay. However, ribosomal pausing can also be employed for regulatory purposes, and controlled translational delays are used to help co-translational folding of the nascent polypeptide on the ribosome, as well as a tactic to delay translation of a protein while its encoding mRNA is being localized within the cell. However, other responses to pausing trigger ribosomal frameshift events. Recent discoveries are thus revealing a wide variety of mechanisms used to respond to translational pausing and thus regulate the flow of ribosomal traffic on the mRNA population.     

Halting a cellular production line: responses to ribosomal pausing during translation

Cellular protein synthesis is a complex polymerization process carried out by multiple ribosomes translating individual mRNAs. The process must be responsive to rapidly changing conditions in the cell that could cause ribosomal pausing and queuing. In some circumstances, pausing of a bacterial ribosome can trigger translational abandonment via the process of trans-translation, mediated by tmRNA (transfer-messenger RNA) and endonucleases. Together, these factors release the ribosome from the mRNA and target the incomplete polypeptide for destruction. In eukaryotes, ribosomal pausing can initiate an analogous process carried out by the Dom34p and Hbs1p proteins, which trigger endonucleolytic attack of the mRNA, a process termed mRNA no-go decay. However, ribosomal pausing can also be employed for regulatory purposes, and controlled translational delays are used to help co-translational folding of the nascent polypeptide on the ribosome, as well as a tactic to delay translation of a protein while its encoding mRNA is being localized within the cell. However, other responses to pausing trigger ribosomal frameshift events. Recent discoveries are thus revealing a wide variety of mechanisms used to respond to translational pausing and thus regulate the flow of ribosomal traffic on the mRNA population.     

Analysis of lincomycin resistance mutations in Escherichia coli.

Summary High level lincomycin resistant strains of Escherichia coli were isolated and screened for altered ribosomal proteins and functions. Amongst 58 strains investigated by electrophoresis one had an altered ribosomal protein S7, another one a mutated L14 and two showed altered L15 proteins.A correlation between these alterations and lincomycin resistant growth could not be demonstrated by genetic analysis for any of the mutants. In vitro, however, extracts from the two L15 mutants were less sensitive to inhibition by the drug. A gene locus (lin ^R) responsible for the lincomycin resistance phenotype was mapped at min 30 of the Escherichia coli chromosome near tyrR; it seems to be identical to the previously described linB locus (Apirion, 1967); however, in contrast to these reports it does not seem to alter any ribosomal function.     

Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium

Many mutations in rpsL cause resistance to, or dependence on, streptomycin and are restrictive (hyperaccurate) in translation. Dependence on streptomycin and hyperaccuracy can each be reversed phenotypically by mutations in either rpsD or rpsE . Such compensatory mutations have been shown to have a ram phenotype (ribosomal ambiguity), increasing the level of translational errors. We have shown recently that restrictive rpsL alleles are also associated with a loss of virulence in Salmonella typhimurium . To test whether ram mutants could reverse this loss of virulence, we have isolated a set of rpsD alleles in Salmonella typhimurium . We found that the rpsD alleles restore the virulence of strains carrying restrictive rpsL alleles to a level close to that of the wild type. Unexpectedly, three out of seven mutant rpsD alleles tested have phenotypes typical of restrictive alleles of rpsL , being resistant to streptomycin and restrictive (hyperaccurate) in translation. These phenotypes have not been previously associated with the ribosomal protein S4. Furthermore, all seven rpsD alleles (four ram and three restrictive) can phenotypically reverse the hyperaccuracy associated with restrictive alleles of rpsL . This is the first demonstration that such compensations do not require that the compensating rpsD allele has a ribosomal ambiguity ( ram ) phenotype.