Role of the Initiation Factors in Escherichia coli Translational Initiation

Gualerzi, Claudio O.; Pon, Cynthia L.; Pawlik, R. T.; Canonaco, Maria A.; Paci, Maurizio; Wintermeyer, Wolfgang

doi:10.1007/978-1-4612-4884-2_36

Cited by 26 publications

(22 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The clustering of these rRNA regions on the platform at the interface between 30S and 50S subunits is consistent with the functional relationships between IF-3 and 30S subunits during initiation complex formation. One function of IF-3 is to stimulate the interaction of mRNA and fMet-tRNA with the 30S subunit (38). The relationship between mRNA and the 3' end of 16S mRNA is well known (39)(40)(41).…”

Section: Discussionmentioning

confidence: 99%

Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

Tapprich

Goss

Dahĺberg

1989

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

A single base was mutated from guanine to adenine at position 791 in 16S rRNA in the Escherichia colimrnB operon on the multicopy plasmid pKK3535. The plasmid-coded rRNA was processed and assembled into 30S ribosomal subunits in E. coli and caused a retardation of cell growth. The mutation affected crucial functional roles of the 30S subunit in the initiation of protein synthesis. The affiity of the mutant 30S subunits for 50S subunits was reduced and the association equilibrium constant for initiation factor 3 was decreased by a factor of 10 compared to wild-type 30S subunits. The interrelationship among the region of residue 790 in 16S rRNA, subunit association, and initiation factor 3 binding during initiation complex formation, as revealed by this study, offers insights into the functional role of rRNA in protein synthesis.Several regions of rRNA have been shown to be involved in the process of translation, primarily regions that are single stranded and highly conserved phylogenetically (1,2). The locations of these sequences in Escherichia coli 16S rRNA have now been placed in three-dimensional models ofthe 30S ribosomal subunit (3, 4). In general there is good agreement between structure and function since many single-stranded highly conserved rRNA regions, proposed to carry out related functions in translation, are clustered in the same area of the subunit (5).One example of a region with a structural placement consistent with its proposed function is the 790 loop of 16S rRNA. In this 9-base loop in the central domain of 16S rRNA, 6 of the bases are universally conserved and 2 additional bases are strongly but not universally conserved (6). The loop has been localized to the platform of the subunit in the area that interfaces with the 50S subunit (3, 4, 7). Several studies have established that many of the bases in the 790 loop are exposed in 30S subunits and inaccessible in 70S ribosomes (8-13). Some of these same studies have implicated this region in the process of subunit association (9-11).The chemical modification studies by Moazed and Noller (13,14), in which rRNA accessibilities were probed in the presence of tRNA or antibiotics, showed that the 790 loop influences several aspects of protein synthesis aside from subunit association. To further define its functional role, the universally conserved guanine at position 791 was mutated to an adenine by site-directed mutagenesis. Here we report on the structural and functional characteristics of the resulting mutant 30S subunits. MATERIALS AND METHODSMutagenesis and Expression. The single-base mutation at position 791 was constructed by oligonucleotide-directed mutagenesis in M13 as described by Zoller and Smith (15).The EcoRI-Xba I fragment from rrnB was cloned into M13mpl9 and the template was isolated from the ung-dutstrain RZ1032 to increase the frequency of mutagenesis (16). After mutagenesis M13 isolates were screened by DNA sequencing (17) and the Bgl II-Xba I fragment containing the mutation was cloned into the expression vector pKK353...

show abstract

Section: Discussionmentioning

confidence: 99%

Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

Tapprich

Goss

Dahĺberg

1989

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Four-factor recombination analysis positioned the inJB gene at 145°on the B. subtilis chromosome, between the poiC and spcB loci.' This location is distinct from those of the other major ribosomal protein and rRNA gene clusters of B. subtilis.The initiation step of protein synthesis has been extensively studied in the gram-negative bacterium Escherichia coli by biochemical (15,17,23) and genetic (7, 16) techniques. Fractionation of E. coli cell extracts has resulted in the identification and purification of three protein synthesis initiation factors (IF1, IF2, and IF3 ) that stimulate the formation of the 30S preinitiation complex.…”

mentioning

confidence: 99%

“…The initiation step of protein synthesis has been extensively studied in the gram-negative bacterium Escherichia coli by biochemical (15,17,23) and genetic (7,16) techniques. Fractionation of E. coli cell extracts has resulted in the identification and purification of three protein synthesis initiation factors (IF1, IF2, and IF3 ) that stimulate the formation of the 30S preinitiation complex.…”

mentioning

confidence: 99%

Isolation and molecular genetic characterization of the Bacillus subtilis gene (infB) encoding protein synthesis initiation factor 2

et al. 1990

View full text Add to dashboard Cite

Western blot (immunoblot) analysis of Bacillus subtilis cell extracts detected two proteins that cross-reacted with monospecific polyclonal antibody raised against Escherichia coli initiation factor 2a (IF2a). Subsequent Southern blot analysis of B. subtils genomic DNA identified a 1.3-kilobase (kb) Hindu fragment which cross-hybridized with both E. coli and Bacillus stearothermophilus IF2 gene probes. This DNA was cloned from a size-selected B. subtilis plasmid library. The cloned HindUI fragment, which was shown by DNA sequence analysis to encode the N-terminal half of the B. subtilis IF2 protein and 0.2 kb of upstream flanking sequence, was utilized as a homologous probe to clone an overlapping 2.76-kb ClaI chromosomal fragment containing the entire IF2 structural gene. The Hindm fragment was also used as a probe to obtain overlapping clones from a Agtll library which contained additional upstream and downstream flanking sequences. Sequence comparisons between the B. subtilis IF2 gene and the other bacterial homologs from E. coli, B. stearothermophilus, and Streptococcus faecium displayed extensive nucleic acid and protein sequence homologies. The B. subtilis injB gene encodes two proteins, IF2a (78.6 kilodaltons) and IF2( (68.2 kilodaltons); both were expressed in B.subtilis and E. coli. These two proteins cross-reacted with antiserum to E. coli IF2a and were able to complement in vivo an E. coli inJB gene disruption. Four-factor recombination analysis positioned the inJB gene at 145°on the B. subtilis chromosome, between the poiC and spcB loci.' This location is distinct from those of the other major ribosomal protein and rRNA gene clusters of B. subtilis.The initiation step of protein synthesis has been extensively studied in the gram-negative bacterium Escherichia coli by biochemical (15,17,23) and genetic (7, 16) techniques. Fractionation of E. coli cell extracts has resulted in the identification and purification of three protein synthesis initiation factors (IF1, IF2, and IF3 ) that stimulate the formation of the 30S preinitiation complex. The 30S'preinitiation complex is composed of a 30S ribosome subunit bound to initiation factors, mRNA, and 'formylmethionyl initiator tRNA (fMet-tRNA'et); a molecule of GTP is bound by IF2. IF2 appears to selectively discriminate in favor of fMet-tRNAfmet binding to the 30S subunit (21). IF3 is involved in additional aspects of the recognition and selection of fMet-tRNAfmt (21). IF3 also may participate in mRNA recognition (14). IF1 stimulates the activities of the other two initiation factors and is required for the selection of initiator tRNA by 70S complexes (21). IF1 and IF3 are released upon formation of a 70S ribosomal initiation complex. This coupling of ribosomal subunits, with the mRNA start codon base paired to the anticodon of fMet-tRNAfMet within the peptidyl site, is essentially irreversible. Hydrolysis of the IF2-bound GTP is apparently concomitant with the release of IF2 from the 70S initiation complex. Release of IF2 leaves a 70S ribosome poised to en...

show abstract

“…The same single-codon gap following proline 164 of the Dsg protein was required in both the M. xanthus and the B. stearothermophilus sequences to align them with their cognate E. coli sequence. Also, the three amino acid sequences have similar hydropathy profiles (8). The pl of the Dsg protein is predicted from its composition to be 10.5; the pIs of E. coli IF3 and B. stearothermophilus IF3 are predicted to be 10.4 and 10.7, respectively.…”

mentioning

confidence: 94%

The dsg gene of Myxococcus xanthus encodes a protein similar to translation initiation factor IF3

1994

View full text Add to dashboard Cite

The dsg mutants of Myxococcus xanthus are defective in fruiting body development and sporulation, yet they grow normally. The deduced amino acid sequence of the dsg gene product is 50 and 51% identical to the amino acid sequence of translation initiation factor IF3 of both Escherichia coli and Bacillus stearothermophilus, respectively. However, the Dsg protein has a carboxy-terminal extension of 66 amino acids, which are absent from its E. coli and B. stearothermophilus homologs. The Shine-Dalgarno sequence GGAGG and 5 bases further upstream are identical in M. xanthus and several enteric bacteria, despite the wide phylogenetic gap between these species. The infC gene, which encodes IF3 in enteric bacteria, starts with the atypical translation initiation codon AUU, which is known to be important for regulating the cellular level of IF3 in E. coli. Translation of the Dsg protein overexpressed from the M. xanthus dsg gene in E. coli cells initiates at an AUC codon, an atypical initiation codon in the AUU class. The dsg mutants DK429 and DK439 carry the same missense mutation that changes Gly-134 to Glu in a region of amino acid identity.

show abstract

Role of the Initiation Factors in Escherichia coli Translational Initiation

Cited by 26 publications

References 28 publications

Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

Isolation and molecular genetic characterization of the Bacillus subtilis gene (infB) encoding protein synthesis initiation factor 2

The dsg gene of Myxococcus xanthus encodes a protein similar to translation initiation factor IF3

Contact Info

Product

Resources

About