Biomedical subjects

M C Ganoza

Publications and source records attributed to M C Ganoza.

At least 19 recordsLinked to original sources

PubMed2006

Molecular localization of a ribosome-dependent ATPase on Escherichia coli ribosomes.

We have previously isolated and described an Escherichia coli ribosome-bound ATPase, RbbA, that is required for protein synthesis in the presence of ATP, GTP and the elongation factors, EF-Tu and EF-G. The gene encoding RbbA, yhih, has been cloned and the deduced protein sequence harbors two ATP-motifs and one RNA-binding motif and is homologous to the fungal EF-3. Here, we describe the isolation and assay of a truncated form of the RbbA protein that is stable to overproduction and purification. Chemical protection results show that the truncated RbbA specifically protects nucleotide A937 on the 30S subunit of ribosomes, and the protected site occurs at the E-site where the tRNA is ejected upon A-site occupation. Other weakly protected bases in the region occur at or near the mRNA binding site. Using radiolabeled tRNAs, we study the stimulating effect of this truncated RbbA on the binding and release of different tRNAs bound to the (aminoacyl) A-, (peptidyl) P- and (exit) E-sites of 70S ribosomes. The combined data suggest plausible mechanisms for the function of RbbA in translation.

Adenosine Triphosphatases↗

PubMed2001

Functional interactions of an Escherichia coli ribosomal ATPase.

The gene encoding ribosome-bound ATPase (RbbA), which occurs bound to 70S ribosomes and 30S subunits, has been identified. The amino-acid sequence of RbbA reveals the presence of two ATP-binding domains in the N-terminal half of the protein. RbbA harbors an intrinsic ATPase activity that is stimulated by both 70S ribosomes and 30S subunits. Here we show that purified recombinant RbbA markedly stimulates polyphenylalanine synthesis in the presence of the elongation factors Tu and G (EF-Tu and EF-G) and that the hydrolysis of ATP by RbbA is required to stimulate synthesis. RbbA is reported to have affinity for EF-Tu but not for EF-G. Additionally, RbbA copurifies with 30S ribosomal subunits and can be crosslinked to the ribosomal protein S1. Studies using a spectrum of antibiotics, including some of similar function, revealed that hygromycin, which binds to the 30S subunit, has a significant effect on the ATPase activity and on the affinity of RbbA for ribosomes. A possible role for RbbA in protein-chain elongation is proposed.

Adenosine Triphosphatases↗

PubMed2001

A ribosomal ATPase is a target for hygromycin B inhibition on Escherichia coli ribosomes.

We demonstrate that the transfer of fully charged aminoacyl-tRNAs into peptides directed by the MS2 RNA template requires both ATP and GTP, initiation factors (IF1, IF2, and IF3), elongation factors (EF-Tu, EF-Ts, and EF-G), and the ribosomal ATPase (RbbA). The nonhydrolyzable analogue AMPPCP inhibits the reactions, suggesting that hydrolysis of ATP is required for synthesis. The RbbA protein occurs bound to ribosomes and stimulates the ATPase activity of Escherichia coli 70S and 30S particles. The gene encoding RbbA harbors four ATP binding domains; the C-terminal half of the protein bears extensive sequence similarity to EF-3, a ribosome-dependent ATPase. Here, we show that the antibiotic hygromycin B selectively inhibits the ATPase activity of RbbA. Other antibiotics with similar effects on miscoding, streptomycin and neomycin, as well as antibiotics that impair peptide bond synthesis and translocation, had little effect on the ATPase activity of RbbA on 70S ribosomes. Immunoblot analysis indicates that at physiological concentrations, hygromycin B selectively releases RbbA from 70S ribosomes. Hygromycin B protects G1494 and A1408 in the decoding region, and RbbA enhances the reactivity of A889 and G890 of the 16S rRNA switch helix region. Cross-linking and X-ray diffraction data have revealed that this helix switch and the decoding region are in close proximity. Mutations in the switch helix (889-890) region affect translational fidelity and translocation. The binding site of hygromycin B and its known dual effect on the fidelity of decoding and translocation suggest a model for the action of this drug on ribosomes.

Adenosine Triphosphatases↗

PubMed2000

Peptide bond synthesis: function of the efp gene product.

The efp gene encodes a protein that is essential for the growth and for the viability of Escherichia coli cells. Interruption of this gene results in cell death due to a defect in protein synthesis. We report here that the EFP protein, encoded by the efp gene, is required for in vitro reconstitution of polypeptide synthesis in a system programmed by a native template which contains each of the purified initiation factors, IF1, IF2, IF3; the elongation factors, EFTu, EFTs and EFG, and a protein called W that is required to eject tRNAs from ribosomes. The EFP protein is required for enhancing the rate and the extent of synthesis in the presence of all of the above factors. The EFP protein stimulates synthesis of poly(Phe) programmed with poly(rU) only if N-acetyl Phe-tRNA initiates the reactions under conditions that foster the dissociation of the 70S ribosome. Study of the ability of the ribosome to synthesize a number of fMet-initiated dipeptides from CCA amino acyl acceptors suggests that EFP acts to promote synthesis with acceptors that are poor donors for the the reconstituted peptidyl transferase.

Bacterial Proteins↗

PubMed1999

Identification of a ribosomal ATPase in Escherichia coli cells.

Eukaryotic ribosomes harbor an ATPase activity that has been shown to be essential for translation elongation in some lower fungi. Here we report the first identification of a ribosome bound ATPase, RbbA, in E. coli cells. RbbA accounts for most of the ATPase activity associated with 70S ribosomes and 30S ribosomal subunits. Both native and recombinant RbbA were purified and shown to possess ribosome-dependent ATPase activities and to stimulate polyphenylalanine synthesis in vitro. Biochemically, RbbA is similar to the fungi-specific translation elongation factor 3 (EF-3) and cross-reacts with antibody raised against EF-3. The gene encoding RbbA is identified as ORF yhih and the predicted RbbA amino acid sequence is 40% similar to that of the C-terminal half of EF-3. The discovery of a ribosomal ATPase in a prokaryotic cell suggests a common, conserved function for these proteins in translation.

Adenosine Triphosphatases↗

PubMed1999

Molecular characterization of a prokaryotic translation factor homologous to the eukaryotic initiation factor eIF4A.

Initiation of translation involves a complex series of reactions that result in the formation of an initiation complex at the proper start site of the mRNA. These reactions, particularly those that involve the binding of the mRNA to the small subunit of the ribosome, are not fully understood. Here we show that one of the factors (W2) required to reconstitute translation in E. coli is encoded by the deaD gene which harbors 87% amino acid sequence similarly to the eukaryotic (eIF4A). Antibodies against the eukaryotic eIF4A cross-react with the E. coli protein. We describe the overexpression of the W2 protein from recombinant clones and its purification in one step by the use of a His tag at the N-terminus of its sequence. We report a rapid assay for the W2 protein that scores for initiation and elongation programmed by a native mRNA template. The W2 protein promotes initiation programmed by the mRNA that harbors secondary structures. The W2 protein is not required in standard initiation assays programmed by synthetic mRNAs of defined sequence that lack this feature. We conclude that W2 is an important factor for initiation in eukaryotic and prokaryotic cells.

Adenosine Triphosphate↗

PubMed1998

The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria.

The oxazolidinones represent a new class of antimicrobial agents which are active against multidrug-resistant staphylococci, streptococci, and enterococci. Previous studies have demonstrated that oxazolidinones inhibit bacterial translation in vitro at a step preceding elongation but after the charging of N-formylmethionine to the initiator tRNA molecule. The event that occurs between these two steps is termed initiation. Initiation of protein synthesis requires the simultaneous presence of N-formylmethionine-tRNA, the 30S ribosomal subunit, mRNA, GTP, and the initiation factors IF1, IF2, and IF3. An initiation complex assay measuring the binding of [3H]N-formylmethionyl-tRNA to ribosomes in response to mRNA binding was used in order to investigate the mechanism of oxazolidinone action. Linezolid inhibited initiation complex formation with either the 30S or the 70S ribosomal subunits from Escherichia coli. In addition, complex formation with Staphylococcus aureus 70S tight-couple ribosomes was inhibited by linezolid. Linezolid did not inhibit the independent binding of either mRNA or N-formylmethionyl-tRNA to E. coli 30S ribosomal subunits, nor did it prevent the formation of the IF2-N-formylmethionyl-tRNA binary complex. The results demonstrate that oxazolidinones inhibit the formation of the initiation complex in bacterial translation systems by preventing formation of the N-formylmethionyl-tRNA-ribosome-mRNA ternary complex.

Acetamides↗

PubMed1997

The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis.

Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A. Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (KanR) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA+ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-KanR gene in the chromosome grew at 33 degrees C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44 degrees C. Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44 degrees C. At 44 degrees C, mutant cells are severely defective in peptide-bond formation. We conclude that the efp gene is essential for cell viability and is required for protein synthesis.

Chromosomes, Bacterial↗

PubMed1997

Molecular characterization of the prokaryotic efp gene product involved in a peptidyltransferase reaction.

The translation factor EF-P is required for efficient prokaryotic peptide bond synthesis on 70S ribosomes from fMet-tRNAfMet. This protein has been purified from Escherichia coli cells and the gene, efp, encoding it has been cloned and sequenced. We have isolated recombinant clones which overexpress a protein that co-migrates with purified EF-P upon SDS-PAGE analysis. Using these clones, we report the purification, crystallization and initial characterization of the efp gene product. The mechanism by which EF-P stimulates peptide-bond synthesis was studied using several antibiotics that inhibit translocation, peptide-bond synthesis and decoding. The stimulation of peptidyltransferase by EF-P was not inhibited by antibiotics that affect translocation and occupation of the A site (in the elongation state), ie thiostrepton, viomycin, neomycin and fusidic acid but was inhibited by streptomycin as well as by inhibitors of peptidyltransferase, chloramphenicol and lincomycin. This observation and the requirement for L16 but not for the L7/L12 nor L6 or L11 r-proteins suggest that the binding site for EF-P may overlap the peptidyltransferase center of the ribosome.

Anti-Bacterial Agents↗

PubMed1996

The ribosome as affinity matrix': efficient purification scheme for translation factors.

A convenient method to purify each of the non-ribosomal proteins required to translate a native mRNA in vitro is described. In this scheme, the ribosome is used as an 'affinity' matrix to selectively elute the non-ribosomal proteins required for translation that are bound to these particles. Different sets of these proteins can be eluted with solutions of Mg2+ and NH4+ of various concentrations from either 70S, or 30S and 50S particles. A scheme for the purification of each initiation, elongation and release factor and 20 aminoacyl-tRNA synthetases is described. Specific examples of the purification of the initiation (IF-1, IF-2, IF-3) and elongation (EF-Tu and EF-G) factors and for a protein called 'rescue', which affects the association of native ribosomal subunits, are given. A scheme for the purification of EF-P, which stimulates peptide-bond synthesis and one of the W proteins, which permit reconstitution of translation is also described. The procedure markedly simplifies the isolation, in homogeneous form, of all the non-ribosomal proteins required to reconstruct translation.

Amino Acyl-tRNA Synthetases↗

PubMed1995

A new factor from Escherichia coli affects translocation of mRNA.

Reconstitution of protein synthesis from purified translation factors on ribosomes from Escherichia coli has revealed the requirement for a protein, W, that affects chain elongation and is essential to reconstitute the process (Ganoza, M. C., Cunningham, C., and Green, R. M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1648-1652). We report that W has no effect on initiation complex formation by 30 or 70 S ribosomes or on the association of ribosomal subunits, peptide bond synthesis, or binding Ala-tRNA, which is the second amino acid of the coat protein of the MS2 RNA virion. W has a pronounced effect on tripeptide synthesis, and is obligatory for the synthesis of the coat protein or of the hexapeptide encoded by f2am3 RNA. Extracts from a temperature-sensitive mutant of the translocase, EF-G, were purified free of the W protein and were used to score for translocation defects. W is required for binding Ser-tRNA, the third N-terminal amino acid of the MS2 or f2 RNA coat protein to ribosomes bearing fMet-Ala-tRNA, as well as for the ejection of deacyl-tRNA from ribosomes, which occurred concomitant with the binding of the Ser-tRNA. We propose that W functions by ejecting tRNAs from ribosomes in a step that precedes the movement of mRNA during translocation.

Bacterial Proteins↗

PubMed1994

Potential secondary structure at the translational start domain of eukaryotic and prokaryotic mRNAs.

In order to identify conserved potential secondary structures within translational start sites, mRNA sequences derived from different species were studied with programs able to depict such features. The potential secondary structure of 71 bases around the initiator AUG or AUGs in the coding sequences of 290 eukaryotic mRNAs was first examined and compared to 290 similarly analyzed regions derived from prokaryotic mRNA sequences (Nucleic Acids Res (1987) 15, 345-360). In both sets of sequences the initiator codon was often found to be in an open potential structure whereas a denser region characterized by nearly-periodic spacings defined the coding regions. Randomization of the sequences obliterated the observed patterns suggesting that the structure of the mRNA may determine these differences. Three sets of eukaryotic and prokaryotic mRNAs of approximately equal length were analyzed and found to preserve an open unpaired non-coding region 5' to the start codon. The start codon was found free of potential secondary structure in over 80% of all the sequences analyzed. These data, and study of mutants that restrict the accessibility of the start codon to the ribosomal initiation complex, suggest that both the prokaryotic and eukaryotic mRNA start sites must occur free of potential secondary structure for efficient initiation. A striking difference of the eukaryotic mRNA sequences analyzed was the high propensity of the coding region vicinal to the start codon to form secondary structures. Certain translation-defective mutants exhibit impaired formation of these secondary structures suggesting that the structure of the coding regions adjacent to the start codons of eukaryotic mRNAs may be an important, thus far unexamined, determinant of initiation. We propose that, for all genes studied, the transition in secondary structure between the coding and non-coding regions may be an important determinant of initiation.

Animals↗

PubMed1993

Reconstruction of translation. Evidence for the involvement of the rescue protein in the association/dissociation of ribosomal subunits.

The in vitro reversal of conditionally lethal mutations has greatly aided the study of translation. N4316 is a mutant of Escherichia coli that has a temperature-sensitive defect in a protein called the rescue protein. Without the rescue protein, translation in vivo and in vitro is drastically reduced and frameshift errors, as well as increased read-through of nonsense codons, occurs. Using reversal of temperature-sensitivity as an assay, the rescue protein was purified from a ribosomal eluate of the parental (D10) strain. Composite polyacrylamide/agarose gel electrophoresis and sedimentation on sucrose density gradients were employed to examine the distribution of 70S ribosomes and ribosomal subunits in the mutant (N4316) and the parental (D10) extracts at restrictive (43 degrees C) and non-restrictive (35 degrees C) temperatures. Fewer polysomes and a larger proportion of 70S ribosomes relative to subunits were observed at 43 degrees C with N4316, but not with D10 extracts. Addition of the rescue protein had no effect at 35 degrees C with either strain, but restored the polysome pattern of N4316 at 43 degrees C. The purified rescue protein labelled by methylation retained activity and bound preferentially to 30S subunits. Rescue bound to 30S particles prevented the action of IF-3 fostering formation of 70S ribosomes. Thus the rescue protein enables formation of 70S ribosomes from 30S and 50S subunits. 70S ribosomes which contain the rescue protein are active in translation and resist dissociation induced by high centrifugal fields. We propose that the rescue protein alters the conformation of 70S ribosomes resulting in a tighter association of subunits which, in turn, fosters both higher rates and increased accuracy of translation.

Amino Acyl-tRNA Synthetases↗

PubMed1992

The asparaginyl-tRNA synthetase gene encodes one of the complementing factors for thermosensitive translation in the Escherichia coli mutant strain, N4316.

Escherichia coli strain N4316 is a mutant that exhibits temperature-sensitive growth at 43 degrees C and temperature-sensitive translation in vivo and in vitro. Extracts of the mutant produce an aberrant pattern of translation products of MS2 bacteriophage RNA. Previous work has shown that a protein, called 'rescue', isolated from the parental strain partly corrects the defective translation in vitro. Here we report the purification to homogeneity of a second factor from ribosomal eluates of the wild-type parental strain; the purified protein is a homodimer of 54 kDa. The partial sequence of the second protein was determined, and a recombinant plasmid was isolated based on its ability to complement the temperature-sensitive growth phenotype of the mutant at the non-permissive temperatures. The cloned gene was sequenced, mapped to the 20.9-min region of the E. coli chromosome and shown to code for a 466-amino-acid protein with a molecular mass of 52 kDa. Analysis of the DNA sequence and the correspondence to that of the partial protein sequence has identified the complementing factor as asparaginyl-tRNA synthetase. Marker rescue experiments indicate that the asnS mutation in N4316 resides within the motif 2 domain of the synthetase. A potential role of this synthetase in restoring normal protein synthesis with respect to ribosomal frameshifting, read-through of nonsense codons and protein copy number is discussed.

Amino Acid Sequence↗

PubMed1992

Effect of deletions 5' to the translation initiation sequence on the expression of an mRNA in animal cells.

To learn if an mRNA.18S rRNA interaction or a special secondary structure in the mRNA start region is essential for translation in eukaryotic cells, we constructed recombinant plasmids with the SV40 early promoter 5' to part of the Escherichia coli tufB-lacZ gene. Deletion of bases potentially complementary to the 18S rRNA highly increased the transient beta-galactosidase expressed in transfected CHO cells. Deletion of bases that fostered formation of potential hairpins with the mRNA 5'-terminus or altered the structure of the coding region reduced beta-galactosidase activity suggesting that these features of the mRNA secondary structure may be essential for initiation of translation. Computer aided analysis of the potential structure of 290 mRNAs suggests these are conserved features of the initiation region.

Animals↗

PubMed1991

Cloning, sequencing and overexpression of the gene for prokaryotic factor EF-P involved in peptide bond synthesis.

A soluble protein EF-P (elongation factor P) from Escherichia coli has been purified and shown to stimulate efficient translation and peptide-bond synthesis on native or reconstituted 70S ribosomes in vitro. Based on the partial amino acid sequence of EF-P, 18- and 24-nucleotide DNA probes were synthesized and used to screen lambda phage clones from the Kohara Gene Bank. The entire EF-P gene was detected on lambda clone #650 which contains sequences from the 94 minute region of the E.coli genome. Two DNA fragments, 3.0 and 0.78 kilobases in length encompassing the gene, were isolated and cloned into pUC18 and pUC19. Partially purified extracts from cells transformed with these plasmids overrepresented a protein which co-migrates with EF-P upon SDS polyacrylamide gel electrophoresis, and also exhibited increased EF-P mediated peptide-bond synthetic activity. Based on DNA sequence analysis of this gene, the EF-P protein consists of 187 amino acids with a calculated molecular weight of 20,447. The sequence and chromosomal location of EF-P establishes it as a unique gene product.

Amino Acid Sequence↗

PubMed1991

A proposed role for IF-3 and EF-T in maintaining the specificity of prokaryotic initiation complex formation.

Initiation factor-free 30S subunits of E. coli ribosomes bind aminoacyl-tRNAs more efficiently than fMet-tRNA(fMet). Elongator-tRNA binding was unaffected by IF-1 or IF-2 but was inhibited by IF-3. Their combination reduced this binding up to 40% and stimulated that of fMet-tRNA(fMet). Unexpectedly, EF-T also prevented elongator-tRNA binding by complexing both to the 30S and to the aminoacyl-tRNAs. Using AUGU3 as mRNA, elongator-tRNAs competed with fMet-fRNA(fMet) and with tRNA(fMet), fMet-tRNA(fMet) reacted with puromycin after addition of 50S subunits suggesting that it occupied the P site. EF-T directed binding of phe-tRNA to the 30S.AUGU3 complex at the A site only if fMet-tRNA(fMet) or tRNA(fMet) filled the P/E site. We propose that one function of EF-T may be to prevent the entry of aminoacyl-tRNAs into the 30S particle during initiation. The possibility that a special site for fMet-tRNA resides on 16S rRNA is also discussed.

Binding Sites↗

PubMed1989

Regulation of gene expression at the translational level. The rescue factor reverses thermosensitive protein synthesis in N4316, a conditionally-lethal mutant of Escherichia coli defective in translation.

Extracts of the conditionally-lethal mutant Escherichia coli N4316 are defective in a newly described translation factor, the rescue protein. We have analyzed the in vitro translation products of this mutant by gel electrophoresis during normal and arrested synthesis at the permissive and non-permissive temperatures. Translation programmed with MS2 bacteriophage RNA at the non-permissive temperature results in highly reduced synthesis of the coat protein with no detectable levels of the maturation and replicase products. Thus the relative number of copies of proteins synthesized by the ribosomes is altered in this mutant. In addition, there is mistranslation of the coat gene which results in the overproduction of the phage encoded no. 7 protein. Aberrant synthesis is also reflected in the increased read-through of termination codons during synthesis directed by phage RNAs harbouring amber mutations in the coat cistron. The rescue protein, purified from the parental strain, is able to complement the thermosensitive defect and restore proper synthesis. Biochemical characterization of the defect in the absence of rescue shows no detectable deficiency in the extent of initiation complex formation in reactions inhibited with sparsomycin. Peptidyltransferase is fully active as judged by the kinetics of formylmethionine-puromycin formation. However, rescue does exert an effect at the level of termination. In addition, the thermolability of the mutant can be reversed by dissociating 70S ribosomes into 30S and 50S subunits. Based on these and other observations, we propose tht rescue mediates a novel function in the association/dissociation of ribosomal subunits which is essential to the accuracy and efficiency of translation.

Bacterial Proteins↗