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C Jain

Publications and source records attributed to C Jain.

At least 19 recordsLinked to original sources
PubMed2002

Identification of multicopy suppressors of the pcnB plasmid copy number defect in Escherichia coli.

Plasmids containing a ColE1 origin of replication are widely used for cloning purposes in Escherichia coli. Among the host factors that affect the copy number of ColE1 plasmids is the E. coli protein poly(A) polymerase I (PAP I), which regulates the intracellular level of RNA I, a ColE1-encoded negative regulator of plasmid replication. In strains that lack PAP I, RNA I levels are elevated, resulting in reduced levels of ColE1 plasmids in the cell. PAP I is encoded by the gene pcnB. We devised a genetic approach, based on the identification of multicopy suppressor clones, to identify trans-acting factors that can help offset the ColE1 plasmid copy number defect in a pcnB (-) genetic background. Using this strategy, we identified suppressors that mapped to two regions of the E. coli chromosome. The suppressor activity of one of the chromosomal regions was localized to the rssB gene, a response regulator gene known to be involved in the turnover of the stationary-phase sigma factor, RpoS. The second suppressor maps to min 55.4 of the E. coli chromosome, and the factor responsible for the suppressor activity appears to be a novel RNA or protein.

Bacterial Proteins↗
PubMed2001

Structural model for the cooperative assembly of HIV-1 Rev multimers on the RRE as deduced from analysis of assembly-defective mutants.

The functional efficacy of the HIV-1 Rev protein is highly dependent on its ability to assemble onto its HIV-1 RNA target (the RRE) as a multimeric complex. To elucidate the mechanism of multimeric assembly, we have devised two rapid and broadly applicable strategies for examining cooperative interactions between proteins bound to RNA, one based on cooperative translational repression of a two-site reporter and the other on gel shift analysis with crude E. coli extracts. Using these strategies, we have identified two distinct surfaces of Rev (head and tail) that are critical for different steps in multimeric assembly. Our data indicate that Rev assembles cooperatively on the RRE via a series of symmetrical tail-to-tail and head-to-head protein-protein interactions. The insights into molecular architecture suggested by these findings have enabled us to derive a structural model for Rev and its multimerization on the RRE.

Allosteric Site↗
PubMed2000

An evolutionarily conserved RNA stem-loop functions as a sensor that directs feedback regulation of RNase E gene expression.

RNase E is a key regulatory enzyme that controls the principal pathway for mRNA degradation in Escherichia coli. The cellular concentration of this endonuclease is governed by a feedback mechanism in which RNase E tightly regulates its own synthesis. Autoregulation is mediated in cis by the 361-nucleotide 5' untranslated region (UTR) of rne (RNase E) mRNA. Here we report the determination of the secondary structure of the rne 5' UTR by phylogenetic comparison and chemical alkylation, together with dissection studies to identify the 5' UTR element that mediates autoregulation. Our findings reveal that the structure and function of the rne 5' UTRs are evolutionarily well conserved despite extensive sequence divergence. Within the rne 5' UTRs are multiple RNA secondary structure elements, two of which function in cis to mediate feedback regulation of rne gene expression. The more potent of these two elements is a stem-loop structure containing an internal loop whose sequence is the most highly conserved of any region of the rne 5' UTR. Our data show that this stem-loop functions as a sensor of cellular RNase E activity that directs autoregulation by modulating the degradation rate of rne mRNA in response to changes in RNase E activity.

5' Untranslated Regions↗
PubMed1997

RNA recognition by the joint action of two nucleolin RNA-binding domains: genetic analysis and structural modeling.

The interaction of nucleolin with a short stem-loop structure (NRE) requires two contiguous RNA-binding domains (RBD 1+2). The structural basis for RNA recognition by these RBDs was studied using a genetic system in Escherichia coli. Within each of the two domains, we identified several mutations that severely impair interaction with the RNA target. Mutations that alter RNA-binding specificity were also isolated, suggesting the identity of specific contacts between RBD 1+2 amino acids and nucleotides within the NRE stem-loop. Our data indicate that both RBDs participate in a joint interaction with the NRE and that each domain uses a different surface to contact the RNA. The constraints provided by these genetic data and previous mutational studies have enabled us to propose a three-dimensional model of nucleolin RBD 1+2 bound to the NRE stem-loop.

Amino Acid Sequence↗
PubMed1997

Models for pairing of IS10 encoded antisense RNAs in vivo.

Antisense regulation of IS10 transposase synthesis is mediated by a small RNA molecule, RNA-OUT which is complementary to the 5' region of the IS10 transposase mRNA, RNA-IN. Pairing between the two species in vivo prevents initiation of RNA-IN translation by steric occlusion of the ribosome binding site. The goal of this work is to develop a mathematical basis for antisense repression in vivo. Thus, by modeling antisense pairing as a biomolecular reaction in vivo, I have developed equations which relate the degree of translation inhibition to a relative pairing rate constant, k, and the in vivo RNA-OUT concentration. Using the methodology developed here, an analysis of mutations in the first three 5' bases of RNA-IN reveals a semi-logarithmic relationship between k and delta G, the estimated change in the free energy of pairing. Such correlations are not observed for mutations at other positions, implicating only the first three 5' bases of RNA-IN in the formation of a pairing nucleus with RNA-OUT. Finally, an analysis of mutations that affect antisense action at a post-nucleation step has been undertaken here and a specific model for how these mutations may affect antisense pairing is discussed.

Base Composition↗
PubMed1996

A structural model for the HIV-1 Rev-RRE complex deduced from altered-specificity rev variants isolated by a rapid genetic strategy.

A broadly applicable genetic strategy was developed for investigating RNA-protein interactions and applied to the HIV-1 Rev protein. By rapidly screening thousands of Rev-RNA interactions in Escherichia coli, we isolated Rev suppressor mutations that alleviated the deleterious effect of mutations in RRE stem-loop IIB, the high affinity RNA-binding site for Rev. All of these suppressor mutations map to a single arginine-deficient face of a Rev alpha-helix, and some alter the binding specificity of the protein, providing genetic evidence for direct contacts between specific Rev amino acids and RNA nucleotides in the RNA complex of Rev. The spatial constraints suggested by these data have enabled us to model the structure of this complex.

Amino Acid Sequence↗
PubMed1995

IS10 antisense control in vivo is affected by mutations throughout the region of complementarity between the interacting RNAs.

Translation of the IS10 transposase mRNA (RNA-IN) is inhibited by antisense pairing with a small IS10 encoded transcript called RNA-OUT. To further characterize IS10 antisense control, an extensive set of mutations in the region of complementarity between RNA-OUT, and its target RNA-IN have been isolated. These mutations have been characterized for their effects on antisense inhibition of transposase gene translation in vivo. Mutations that confer the strongest defects on translational inhibition are found in the region corresponding to the 5' end of RNA-IN. However, mutations throughout the complementary region affect antisense control regardless of whether mutations are present in RNA-IN alone or as complementary mutations in both RNAs. An analysis of the data presented here suggests that in vivo pairing rates for the wild-type antisense species are very close to being optimal. Some of the motifs found in antisense molecules that may be associated with efficient pairing rates are discussed.

Base Sequence↗
PubMed1995

RNase E autoregulates its synthesis by controlling the degradation rate of its own mRNA in Escherichia coli: unusual sensitivity of the rne transcript to RNase E activity.

RNase E is a key regulatory enzyme that appears to control the principal pathway for mRNA degradation in Escherichia coli. Here, we show that RNase E represses its own synthesis by reducing the cellular concentration of the rne (RNase E) gene transcript. Autoregulation is achieved by modulating the longevity of this 3.6-kb mRNA, whose half-life ranges from < 40 sec to > 8 min depending on the level of RNase E activity in the cell. Feedback regulation is mediated in cis by the 5'-terminal 0.44-kb segment of rne mRNA, which is sufficient to confer this property onto a heterologous transcript to which it is fused. Like the intact protein, an amino-terminal fragment of RNase E lacking 563 amino acid residues can act in trans to repress rne gene expression. Paradoxically, raising the rne gene copy number 21-fold in E. coli causes an unexpected reduction in the concentration of the full-length rne transcript, yet results in a small increase in RNase E protein production. These surprising phenomena are explained in terms of a model in which the degradation of this long and highly labile mRNA commences before elongation of the nascent transcript has been completed. In such circumstances, gene expression can be unusually sensitive to changes in mRNA stability.

Base Sequence↗
PubMed1995

Autoregulation of RNase E synthesis in Escherichia coli.

RNase E plays a central role in controlling mRNA degradation in E. coli. We have investigated the mechanism of RNase E autoregulation. Our data indicate that RNase E autoregulates its synthesis by controlling the decay rate of its own transcript (rne mRNA), which is unusually sensitive to the level of cellular RNase E activity. Feedback regulation is mediated in cis by the rne 5' untranslated region (5' UTR), which can confer this property onto heterologous mRNAs to which it is fused. The marked sensitivity of rne mRNA to regulation by RNase E is also due in part to the susceptibility of nascent rne transcripts to RNase E-mediated degradation.

Cloning, Molecular↗
PubMed1993

New improved lacZ gene fusion vectors.

New plasmid vectors suitable for creating fusions with the lacZ gene have been developed. These vectors represent an improvement over currently available vectors and possess the following features: (1) an undetectable background beta-galactosidase (beta Gal) activity in the absence of fusion, (2) an extended multiple cloning site (MCS), and (3) the ability to conveniently subclone in any one of three translational frames. Medium- and high-copy-number versions of these vectors have been developed.

Base Sequence↗
PubMed1993

IS10 mRNA stability and steady state levels in Escherichia coli: indirect effects of translation and role of rne function.

Translation of the IS10 transposase gene is known to be very infrequent. We have identified mutations whose genetic properties suggest that they act directly to increase or decrease the intrinsic level of translation initiation. Also, we have analysed in detail the effects of these mutations on IS10 mRNA using one particular IS10 derivative. In this case, increases or decreases in translation are accompanied by increases or decreases in both the steady state level and the half-life of transposase mRNA; effects on steady state levels are much more dramatic than effects on message half-life. At wild-type levels of translation initiation, the rate-limiting step in physical decay of full length IS10 message for a particular IS10 derivative is shown to be rne-dependent endonucleolytic cleavage; 3' exonucleases appear to play a secondary role, degrading primary cleavage products. Analysis of interplay between translation mutations and rne function, together with the above observations, suggests that translation stabilizes messages in a general way against rne-dependent endonucleolytic cleavage, and that significant protection may be conferred by one or a few ribosomes. However, dramatic effects of translation on steady state message levels are still observed in an rne mutant and involve the 3' end of the transcript; we propose that these additional effects reflect translation-mediated stimulation of transcript release.

Bacterial Proteins↗
PubMed1993

Preferential cis action of IS10 transposase depends upon its mode of synthesis.

A number of bacterial DNA-binding proteins, including IS element transposases, act preferentially in cis. We show below that the degree of preferential cis action by IS10 transposase depends upon its mode of synthesis at steps subsequent to transcription initiation. Cis preference is increased several fold by mutations that decrease translation initiation, by the presence of IS10-specific antisense RNA and by plasmids that increase the level of cellular RNases. Conversely, cis preference is decreased by mutations that increase translation initiation; in some cases, cis preference is nearly abolished. Mutations that alter the rate of transcription initiation have no effect. In light of other observations, we suggest that cis preference is strongly dependent upon the rate at which transcripts are released from their templates and/or the half-life of the transposase message. These observations provide further evidence that inefficient translation plays multiple roles in the biology of IS10.

Bacterial Proteins↗
PubMed1985

Attitudes of Asian patients in Birmingham to general practitioner services.

Attitudes of Asian patients to the delivery of primary health care in two Birmingham general practices were investigated by questionnaires administered by an Asian ethnic minorities worker who spoke dialects appropriate to the population under investigation. One practice was staffed by Asian doctors the other by British doctors. The responses to the questionnaires were analysed with reference to religion - Sikh, Hindu and Muslim and to the two practices. Choice of doctor appears to be determined more by the proximity of the patient's home to the practice premises than by ethnic considerations. Reported failures to meet the special needs of Asian patients were those inherent in the difficulties of British general practice and were not peculiar to Asian patients. The need for help from an interpreter did not seem to be important.

Adult↗