Biomedical subjects

F Abdulkarim

Publications and source records attributed to F Abdulkarim.

6 recordsLinked to original sources

PubMed2002

Origin-specific reduction of ColE1 plasmid copy number due to mutations in a distinct region of the Escherichia coli RNA polymerase.

Mutations affecting a region of the Escherichia coli RNA polymerase have been isolated that specifically reduce the copy number of ColE1-type plasmids. The mutations, which result in a single amino acid alteration (G1161R) or a 41-amino acid deletion (Delta1149-1190) are located near the 3'-terminal region in the rpoC gene, which encodes the largest subunit (beta ') of the RNA polymerase. The rpoC deletion and the point mutation cause over 20- and 10-fold reductions, respectively, in the copy number of ColE1. ColE1 plasmid numbers are regulated by two plasmid-encoded RNAs: RNA II, which acts as a preprimer for the DNA polymerase I to start initiation of replication, and RNA I, its antisense inhibitor. Altered expression from the RNA I and RNA II promoters in vivo was observed in the RNA polymerase mutants. The RNA I/RNA II ratio is higher in the mutants than in the wild-type strain and this is most probably the main reason for the reduction in the ColE1 copy number in the two rpoC mutants.

Amino Acid Substitution↗

PubMed1996

Homologous recombination between the tuf genes of Salmonella typhimurium.

The genes coding for the translation factor EF-Tu, tufA and tufB are separated by over 700 kb on the circular chromosome of Salmonella typhimurium. The coding regions of these genes have 99% identity at the nucleotide level in spite of the presumed ancient origin of the gene duplication. Sequence comparisons between S. typhimurium and Escherichia coli suggest that within each species the two tuf genes are evolving in concert. Here we show that each of the S. typhimurium tuf genes can transfer genetic information to the other. In our genetic system the transfers are seen as non-reciprocal, i.e. as gene conversion events. However, the mechanism of recombination could be reciprocal, with sister chromosome segregation and selection leading to the isolation of a particular class of recombinant. The amount of sequence information transferred in individual recombination events varies, but can be close to the entire length of the gene. The recombination is RecABCD-dependent, and is opposed by MutSHLU mismatch repair. In the wild-type, this type of recombination occurs at a rate that is two or three orders of magnitude greater than the nucleotide substitution rate. The rate of recombination differs by six orders of magnitude between a recA and a mutS strain. Mismatch repair reduces the rate of this recombination 1000-fold. The rate of recombination also differs by one order of magnitude depending on which tuf gene is donating the sequence selected for. We discuss three classes of model that could, in principle, account for the sequence transfers: (1) tuf mRNA mediated recombination; (2) non-allelic reciprocal recombination involving sister chromosomes; (3) non-allelic gene conversion involving sister chromosomes, initiated by a double-strand break close to one tuf gene. Although the mechanism remains to be determined, the effect on the bacterial cells is tuf gene sequence homogenisation. This recombination phenomenon can account for the concerted evolution of the tuf genes.

Base Sequence↗

PubMed1996

Mutants of EF-Tu defective in binding aminoacyl-tRNA.

Five single amino acid substitution variants of EF-Tu from Salmonella typhimurium were tested for their ability to promote poly(U)-translation in vitro. The substitutions are Leu120 Gln, Gln124 Arg and Tyr160 (Asp or Asn or Cys). They were selected by their kirromycin resistant phenotypes and all substitutions are in domain I at the interface between domains I and III of the EF-Tu.GTP configuration. The different EF-Tu variants exhibit a spectrum of phenotypes. First, k cat/K(M) for the interaction between ternary complex and the programmed ribosome is apparently reduced by the substitutions Leu120 Gln, Gln124 Arg and Tyr160 Cys. Second, this reduction is caused by a defect in the interaction between these EF-Tu variants and aminoacyl-tRNA during translation. Third, in four cases out of five the affinity of the complex between EF-Tu.GTP and aminoacyl-tRNA is significantly decreased. The most drastic reduction is observed for the Gln124 Arg change, where the association constant is 30-fold lower than in the wild-type case. Fourth, missense errors are increased as well as decreased by the different amino acid substitutions. Finally, the dissociation rate constant (kd) for the release of GDP from EF-Tu is increased 6-fold by the Tyr160 Cys substitution, but remains unchanged in the four other cases. These results show that the formation of ternary complex is sensitive to many different alterations in the domain I-III interface of EF-Tu.

Guanosine Triphosphate↗

PubMed1994

Mutations to kirromycin resistance occur in the interface of domains I and III of EF-Tu.GTP.

The antibiotic kirromycin inhibits protein synthesis by binding to EF-Tu and preventing its release from the ribosome after GTP hydrolysis. We have isolated and sequenced a collection of kirromycin resistant tuf mutations and identified thirteen single amino acid substitutions at seven different sites in EF-Tu. These have been mapped onto the 3D structures of EF-Tu.GTP and EF-Tu.GDP. In the active GTP form of EF-Tu the mutations cluster on each side of the interface between domains I and III. We propose that this domain interface is the binding site for kirromycin.

Amino Acids↗

PubMed1991

Missense substitutions lethal to essential functions of EF-Tu.

We have used a simple selection and screening method to isolate function defective mutants of EF-Tu. From 28 mutants tested, 12 different missense substitutions, individually lethal to some essential function of EF-Tu, were identified by sequencing. In addition we found a new non-lethal missense mutation. The frequency of isolation of unique mutations suggests that this method can be used to easily isolate many more. The lethal mutations occur in all three structural domains of EF-Tu, but most are in domain II. We aim to use these mutants to define functional domains on EF-Tu.

Base Sequence↗

PubMedDate not supplied

tRNA-ribosome interactions.

Direct measurements of the rates of dissociation of dipeptidyl-tRNA from the ribosome show that hyperaccurate SmP and SmD ribosomes have unstable A-site binding of peptidyl-tRNA, while P-site binding is extremely stable in relation to the wild type. Error-prone Ram ribosomes, on the other hand, have stable A-site and unstable P-site binding of peptidyl-tRNA. At least for these mutant ribosomes, we conclude that stabilization of peptidyl-tRNA in one site destabilizes binding in the other. Elongation factor Tu (EF-Tu) undergoes a dramatic structural transition from its GDP-bound form to its active GTP-bound form, in which it binds aa-tRNA (aminoacyl-tRNA) in ternary complex. The effects of substitution mutations at three sites in domain I of EF-Tu, Gln124, Leu120, and Tyr160, all of which point into the domain I-domain III interface in both the GTP and GDP conformations of EF-Tu, were examined. Mutations at each position cause large reductions in aa-tRNA binding. An attractive possibility is that the mutations alter the domain I-domain III interface such that the switching of EF-Tu between different conformations is altered, decreasing the probability of aa-tRNA binding. We have previously found that two GTPs are hydrolyzed per peptide bond on EF-Tu, the implication being that two molecules of EF-Tu may interact on the ribosome to catalyze the binding of a single aa-tRNA to the A-site. More recently we found that ribosomes programmed with mRNA constructs other than poly(U), including the sequence AUGUUUACG, invariably use two GTPs per peptide bond in EF-Tu function. Other experiments measuring the protection of aa-tRNA from deacylation or from RNAse A attack show that protection requires two molecules of EF-Tu, suggesting an extended ternary complex. To remove remaining ambiguities in the interpretion of these experiments, we are making direct molecular weight determinations with neutron scattering and sedimentation-diffusion techniques.

Bacterial Proteins↗