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Biomedical subjects

R Karimi

Publications and source records attributed to R Karimi.

15 recordsLinked to original sources
PubMed2000

Perforin lytic activity is controlled by calreticulin.

The components within cytotoxic lymphocyte granules are responsible for a significant fraction of T and NK cell-mediated death. Perforin is stored in these granules together with calreticulin. Calreticulin has long been recognized as a chaperone protein of the endoplasmic reticulum (ER) and is the only resident ER protein to be found in the cytotoxic granules. Here we implicate a role for calreticulin in killing and report that it controls osmotic lysis mediated by purified perforin. Calreticulin, at a concentration of 2.2 x 10-7 M, completely blocked perforin-mediated lysis. Inhibition was stable and held over 5 h. Recombinant calreticulin, at a concentration of 8. 8 x 10-7 M, also blocked lysis, indicating the inhibition was due to calreticulin and not a copurifying protein in the native calreticulin preparations. Using calreticulin domain fragments (expressed as GST fusion proteins), we found inhibitory activity in the high-capacity calcium-binding C-domain, which does not bind perforin. The N- or P-domains, which can bind perforin, were unable to block lysis. The inhibition of lysis was independent of granzyme inactivation or the ability of calreticulin to sequester calcium. Our data indicate that calreticulin regulation of perforin-mediated lysis probably occurs without direct interaction with perforin. We propose a novel model in which calreticulin stabilizes membranes to prevent polyperforin pore formation.

Animals↗
PubMed1999

Shutdown in protein synthesis due to the expression of mini-genes in bacteria.

Mutants of Escherichia coli partially deficient in peptidyl-tRNA hydrolase are killed by the expression of certain very short open reading frames (mini-genes), encoded by the wild-type bar regions of phage lambda. According to the current hypothesis, protein synthesis is shut off, and the host cells die, after essential tRNA species become sequestered due to abnormal translation termination (drop-off) of mini-gene-encoded peptides as peptidyl-tRNA. Here we study variants of bar mini-genes, both in vivo and in vitro, in order to identify the structural elements that influence this inhibition of protein synthesis. Three parameters were measured during the expression of these variants: the rates of normal translation termination, peptidyl-tRNA dissociation from the ribosome and hydrolysis of peptidyl-tRNA by peptidyl-tRNA hydrolase were measured. Previous observations that RRF, EF-G and RF3 stimulated drop-off were confirmed and extended; stimulation by these factors can reach 30-fold. Both factor-stimulated and spontaneous drop-off depended on the nature of the stop signal. The degree of inhibition of cell growth following induction of mini-gene expression could be accounted for in terms of a toxicity index comprising the three parameters above. Inhibition was greatly reduced in cells lacking RF3. Mini-genes with more efficient Shine/Dalgarno sequences killed cells even with normal peptidyl-tRNA hydrolase activity. It is proposed that the retranslation by ribosomes of mini-gene transcripts with efficient ribosome binding (Shine/Dalgarno) sequences strongly contributes to the inhibitory effects of mini-gene expression on protein synthesis.

Bacterial Proteins↗
PubMed1999

Novel roles for classical factors at the interface between translation termination and initiation.

The pathway of bacterial ribosome recycling following translation termination has remained obscure. Here, we elucidate two essential steps and describe the roles played by the three translation factors EF-G, RRF, and IF3. Release factor RF3 is known to catalyze the dissociation of RF1 or RF2 from ribosomes after polypeptide release. We show that the next step is dissociation of 50S subunits from the 70S posttermination complex and that it is catalyzed by RRF and EF-G and requires GTP hydrolysis. Removal of deacylated tRNA from the resulting 30S:mRNA:tRNA posttermination complex is then necessary to permit rapid 30S subunit recycling. We show that this step requires initiation factor IF3, whose role was previously thought to be restricted to promoting specific 30S initiation complex formation from free 30S subunits.

Acetylation↗
PubMed1998

Initiation factors IF1 and IF2 synergistically remove peptidyl-tRNAs with short polypeptides from the P-site of translating Escherichia coli ribosomes.

A novel function of initiation factors IF1 and IF2 in Escherichia coli translation has been identified. It is shown that these factors efficiently catalyse dissociation of peptidyl-tRNAs with polypeptides of different length from the P-site of E. coli ribosomes, and that the simultaneous presence of both factors is required for induction of drop-off. The factor-induced drop-off occurs with both sense and stop codons in the A-site and competes with peptide elongation or termination. The efficiency with which IF1 and IF2 catalyse drop-off decreases with increasing length of the nascent polypeptide, but is quite significant for hepta-peptidyl-tRNAs, the longest polypeptide chains studied. In the absence of IF1 and IF2 the rate of drop-off varies considerably for different peptidyl-tRNAs, and depends both on the length and sequence of the nascent peptide. Efficient factor-catalysed drop-off requires GTP but not GTP hydrolysis, as shown in experiments without guanine nucleotides, with GDP or with the non-cleavable analogue GMP-PNP.Simultaneous overexpression of IF1 and IF2 in vivo inhibits cell growth specifically in some peptidyl-tRNA hydrolase deficient mutants, suggesting that initiation factor-catalysed drop-off of peptidyl-tRNA can occur on a significant scale in the bacterial cell. Consequences for the bacterial physiology of this previously unknown function of IF1 and IF2 are discussed.

Carboxylic Ester Hydrolases↗
PubMed1998

Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome.

Peptidyl-tRNA dissociation from ribosomes is an energetically costly but apparently inevitable process that accompanies normal protein synthesis. The drop-off products of these events are hydrolysed by peptidyl-tRNA hydrolase. Mutant selections have been made to identify genes involved in the drop-off of peptidyl-tRNA, using a thermosensitive peptidyl-tRNA hydrolase mutant in Escherichia coli. Transposon insertions upstream of the frr gene, which encodes RF4 (ribosome release or recycling factor), restored growth to this mutant. The insertions impaired expression of the frr gene. Mutations inactivating prfC, encoding RF3 (release factor 3), displayed a similar phenotype. Conversely, production of RF4 from a plasmid increased the thermosensitivity of the peptidyl-tRNA hydrolase mutant. In vitro measurements of peptidyl-tRNA release from ribosomes paused at stop signals or sense codons confirmed that RF3 and RF4 were able to stimulate peptidyl-tRNA release from ribosomes, and showed that this action of RF4 required the presence of translocation factor EF2, known to be needed for the function of RF4 in ribosome recycling. When present together, the three factors were able to stimulate release up to 12-fold. It is suggested that RF4 may displace peptidyl-tRNA from the ribosome in a manner related to its proposed function in removing deacylated tRNA during ribosome recycling.

Bacterial Proteins↗
PubMed1996

Dissociation rates of peptidyl-tRNA from the P-site of E.coli ribosomes.

We studied the dissociation rates of peptidyl-tRNA from the P-site of poly(U)-programmed wild-type Escherichia coli ribosomes, hyperaccurate variants altered in S12 (SmD, SmP) and error-prone variants (Ram) altered in S4 or S5. The experiments were carried out in the presence and absence of streptomycin, and the effects of neomycin were tested in the wild-type ribosomes. Binding of peptidyl-tRNA to the P-site of wild-type ribosomes is much stronger than to their A-site. Addition of streptomycin dramatically reduces its affinity for the P-site. The S12 alternations make the P-site binding of peptidyl-tRNA much tighter, and the S4, S5 alterations make it weaker than in the case of the wild-type. We find that when binding of peptidyl-tRNA to the A-site is weak, then the affinity for the P-site is stronger, and vice versa. From these results, we formulate a hypothesis for the actions of streptomycin and neomycin based on deformations of the 16S rRNA tertiary structure. The results are also used to interpret some in vivo experiments on translational processivity.

Amino Acid Sequence↗
PubMed1994

Dissociation rate of cognate peptidyl-tRNA from the A-site of hyper-accurate and error-prone ribosomes.

The binding stability of the aminoacyl-tRNA site (A-site), estimated from the dissociation rate constant kd, of AcPhe-Phe-tRNA(Phe) has been studied for wild-type (wt), for hyperaccurate ribosomes altered in S12 [streptomycin-dependent (SmD) and streptomycin-pseudodependent (SmP) phenotypes], for error-prone ribosomes altered in S4 (Ram phenotype), and for ribosomes in complex with the error-inducing aminoglycosides streptomycin and neomycin. The AcPhe2-tRNA stability is slightly and identically reduced for SmD and SmP phenotypes in relation to wt ribosomes. The stability is increased (kd is reduced) for Ram ribosomes to about the same extent as the proof-reading accuracy is decreased for this phenotype. kd is also reduced by the action of streptomycin and neomycin, but much less than the reduction in proof-reading accuracy induced by streptomycin. Similar kd values for SmD and SmP ribosomes indicate that the cause of streptomycin dependence is not excessive drop-off of peptidyl-tRNAs from the A-site.

Binding Sites↗
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↗