Biomedical subjects

Sankar Adhya

Publications and source records attributed to Sankar Adhya.

34 records · Page 2Linked to original sources

PubMed2004

A mutant spacer sequence between -35 and -10 elements makes the Plac promoter hyperactive and cAMP receptor protein-independent.

To determine whether the spacer region between the -35 and -10 elements plays any sequence-specific role, we randomized the GC-rich sequence ((-20)CCGGCTCG(-13)) within the spacer region of the cAMP-dependent lac promoter and selected an activator-independent mutant, which showed extraordinarily high intrinsic activity. The hyperactive promoter is obtained by incorporation of a specific 10-bp-long AT-rich DNA sequence within the spacer, referred to as the -15 sequence, which must be juxtaposed to the upstream end of the -10 sequence for the hyperactivity. The transcription enhancement functions only in the presence of a -35 element. The spacer sequence enhanced both RNA polymerase binding and open complex formation. Isolated in the lac promoter, it also enhanced transcription when placed at two other unrelated promoters. Sequence analysis shows a low GC content and an abundance of stereochemically flexible TG:CA and TA:TA dimeric steps in the -18/-9 region and a strong correlation between the presence of flexible dimeric steps in this region and the intrinsic strength of the promoter.

DNA↗

PubMed2004

An unsubstituted C2 hydrogen of adenine is critical and sufficient at the -11 position of a promoter to signal base pair deformation.

The conserved A:T base pair at the -11 position of the promoters in Escherichia coli is very sensitive to substitutions. In vitro transcription with the galP1 promoter having a natural or unnatural base in either strand at position -11 showed that only a purine base with no side group at C2 in the nontemplate strand is transcriptionally potent; neither a purine with an amino group at C2 nor a pyrimidine support transcription. The amino group at C6 in the omnipresent adenine at -11 does not play any role in promoting transcription. The nature of the base, complementary or noncomplementary, at -11 in the template strand also does not influence transcription. We propose that the adenine, by becoming extrahelical, interacts with an amino acid(s) of the 2.3-2.4 region of sigma for which an unsubstituted C2 hydrogen is critical.

Adenine↗

PubMed2004

Asynchronous basepair openings in transcription initiation: CRP enhances the rate-limiting step.

The mechanism of isomerization (basepair openings) during transcription initiation by RNA polymerase at the galP1 promoter of Escherichia coli was investigated by 2-aminopurine (2,AP) fluorescence. The fluorescence of 2,AP is quenched in DNA duplex and enhanced when the basepair is distorted or deformed. The increase of 2,AP fluorescence was used to monitor basepair distortion at several individual positions in the promoter. We observed that basepair distortions during isomerization are a multi-step process. Three distinct hitherto unresolved steps in kinetic terms were observed, where significant fluorescence change occurs: a fast step with a half-life of around 1 s, which is followed by two slower steps occurring with a half-life in the range of minutes at 25 degrees C. Contrary to commonly held expectations, basepairs at different positions opened by 2,AP assays without any obvious pattern, suggesting that basepair opening is an asynchronous multi-step process. cAMP.CRP, which activates transcription at galP1, enhanced the rate-limiting step.

2-Aminopurine↗

PubMed2004

Axiom of determining transcription start points by RNA polymerase in Escherichia coli.

To investigate the determining factors in the selection of the transcription start points (tsp) by RNA polymerase of Escherichia coli, we systematically deleted or substituted single base pairs (bps) at 25 putative critical positions in the two extended -10 promoters, P1 and P2, of the gal operon. These changes extend downstream from -24 to +1 of the P1 promoter. In vitro transcription assays using supercoiled DNA templates revealed a preference for a purine in the non-template strand for tsp in both promoters. The optimal tsp is the 11th bp counting downstream from the -10 position. A single bp deletion anywhere from -10 to +1 switched the tsp to the next available purine 2-3 bp downstream on the non-template strand whereas deleting a single bp at position from -24 to -11 did not affect the tsp. The nature of the 10 bp sequence of the -10 to -1 region, while affecting promoter strength, did not influence tsp. The cAMP-CRP complex, which stimulates P1 and represses P2, did not affect the tsp selection process. The rules of tsp selection by RNA polymerase containing sigma70 in gal and pyr promoters discussed here may be applicable to others.

Base Sequence↗

PubMed2003

High-density functional display of proteins on bacteriophage lambda.

We designed a bacteriophage lambda system to display peptides and proteins fused at the C terminus of the head protein gpD of phage lambda. DNA encoding the foreign peptide/protein was first inserted at the 3' end of a DNA segment encoding gpD under the control of the lac promoter in a plasmid vector (donor plasmid), which also carried lox P(wt) and lox P(511) recombination sequences. Cre-expressing cells were transformed with this plasmid and subsequently infected with a recipient lambda phage that carried a stuffer DNA segment flanked by lox P(wt) and lox P(511) sites. Recombination occurred in vivo at the lox sites and Amp(r) cointegrates were formed. The cointegrates produced recombinant phage that displayed foreign protein fused at the C terminus of gpD. The system was optimised by cloning DNA encoding different length fragments of HIV-1 p24 (amino acid residues 1-72, 1-156 and 1-231) and the display was compared with that obtained with M13 phage. The display on lambda phage was at least 100-fold higher than on M13 phage for all the fragments with no degradation of displayed products. The high-density display on lambda phage was superior to that on M13 phage and resulted in selective enrichment of epitope-bearing clones from gene-fragment libraries. Single-chain antibodies were displayed in functional form on phage lambda, strongly suggesting that correct disulphide bond formation takes place during display. This lambda phage display system, which avoids direct cloning into lambda DNA and in vitro packaging, achieved cloning efficiencies comparable to those obtained with any plasmid system. The high-density display of foreign proteins on bacteriophage lambda should be extremely useful in studying low-affinity protein-protein interactions more efficiently compared to the M13 phage-based system.

Bacteriophage M13↗

PubMed2003

"Antiparallel" DNA loop in gal repressosome visualized by atomic force microscopy.

DNA looping is often involved in positive and negative regulation of gene transcription in both prokaryotes and eukaryotes. The transcription of the gal operon of Escherichia coli from two overlapping promoters P1 and P2 is negatively regulated via Gal repressosome assembly. It involves binding of two dimeric Gal repressor proteins (GalR) to two operators, O(E) and O(I), flanking the two promoters, and formation of 113 bp DNA loop due to tetramerization of the two bound GalR dimers. The process requires negatively supercoiled DNA and the presence of the histone-like protein HU. Previous modeling of the repressosome based on evaluation of DNA elastic energy suggested a mutual antiparallel, rather than parallel, orientation of the two gal operators in an under-twisted DNA loop. To visualize the Gal loop by atomic force microscopy (AFM), plasmid DNA molecules were constructed with increased distance between the two operators. The AFM results demonstrated the formation of an antiparallel DNA loop in the Gal repressosome consistent with our earlier hypothesis. Importantly, the overall shape of the GalR mediated loop proved to be indistinguishable from that in the chimerical loop of the same size containing two lac operators (instead of two gal operators) and formed by LacI. In addition, a possibility of the gal operon repression mediated by GalR in the absence of HU was shown in the new DNA constructs. Implications of these findings for the DNA structural organization in bacterial nucleoid are discussed.

Bacterial Proteins↗

PubMed2003

Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping.

The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and/or looping. Gal repressor (GalR) is thought to repress transcription from the two promoters of the gal operon of Escherichia coli by forming a DNA loop of approximately 40 nm of DNA that encompasses the promoters. Associated evidence of a topological regulatory mechanism of the transcription repression is the requirement for a supercoiled DNA template and the histone-like heat unstable nucleoid protein (HU). By using single-molecule manipulations to generate and finely tune tension in DNA molecules, we directly detected GalR/HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence. The factors required for gal DNA looping in single-molecule experiments (HU, GalR and DNA supercoiling) correspond exactly to those necessary for gal repression observed both in vitro and in vivo. Our single-molecule experiments revealed that negatively supercoiled DNA, under slight tension, denatured to facilitate GalR/HU-mediated DNA loop formation. Such topological intermediates may operate similarly in other multiprotein complexes of transcription, replication, and recombination.

DNA, Superhelical↗

PubMed2003

Effect of varying the supercoiling of DNA on transcription and its regulation.

The effect of superhelicity of DNA templates on transcription is well documented in several cases. However, the amount of supercoiling that is needed to bring about any changes and the steps at which such effects are exerted were not systematically studied. We investigated the effect of DNA supercoiling on transcription from a set of promoters present on a plasmid by using a series of topoisomers with different superhelical densities ranging from totally relaxed to more than physiological. In vitro transcription assays with these topoisomers in the absence and presence of gene regulatory proteins showed that the effect of negative supercoiling on intrinsic transcription varies from promoter to promoter. Some of those promoters, in which DNA superhelicity stimulated transcription, displayed specific optima of superhelical density while others did not. The results also showed that the amounts of abortive RNA synthesis from two of the promoters decreased and full-length RNA increased with increasing supercoiling, indicating for the first time an inverse relationship between full-length and abortive RNA synthesis and supporting a role of DNA superhelicity in promoter clearance. DNA supercoiling might also influence the point of RNA chain termination. Furthermore, the effect of varying the amount of supercoiling on the action of gene regulatory proteins suggested the mode of action, which is consistent with previous results. Our results underscore the importance of DNA supercoiling in fine-tuning promoter activities, which should be relevant in cell physiology given that local changes in chromosomal supercoiling must occur in different environments.

Animals↗

PubMed2003

Suboperonic regulatory signals.

In prokaryotes, the genome is necessarily small in size, thus creating challenges for gene regulation. Adhya discusses how polycistronic operons can be regulated at the suboperonic level to allow genes to be independently regulated within an operon. This permits the cells to respond to different environmental conditions and allows the genes within operons to encode proteins involved in divergent cellular processes and still be regulated according to the cell's needs. Suboperonic control leads to discoordinate gene expression and can occur through transcriptional regulatory events or translational regulatory events mediated by proteins or cis- or trans-acting RNAs.

Gene Expression Regulation, Bacterial↗

PubMed2002

Operator-bound GalR dimers close DNA loops by direct interaction: tetramerization and inducer binding.

The assembly of the Gal repressosome, a higher order nucleoprotein complex that represses transcription of the gal operon in Escherichia coli, involves the formation of a DNA loop encompassing the promoter segment. GalR dimers bound to two spatially separated operators, O(E) and O(I), specifically interact with the histone-like protein HU and close the loop in supercoiled DNA. We isolated and characterized a GalR mutant containing an amino acid substitution (R282L) that can repress transcription in the absence of HU and supercoiled DNA both in vivo and in vitro. Repression involves the same DNA looping; deletion of either O(E) or O(I) makes the mutant GalR ineffective in repression. This and other results suggest that the R282L substitution increases the normal affinity between two DNA-bound GalR dimers, allowing looping. We conclude that GalR dimers interact directly and do not use HU as an adaptor in loop closure; HU and DNA supercoiling act in concert to stabilize the GalR tetramer. The stronger GalR-GalR interaction also made the gal transcription non-inducible, suggesting that the inducer binding acts by modulating tetramerization.

DNA, Bacterial↗

PubMed2002

Genetic analysis of GalR tetramerization in DNA looping during repressosome assembly.

The Gal repressosome is a nucleoprotein complex consisting of 2 GalR dimers, 1 HU, and 1 DNA loop, which represses the transcription of the gal operon. We have adopted a structure-based genetic approach to complement ongoing physical studies of the complex. Homology-based and subsequent alanine-scanning mutageneses suggest that five residues in the DNA-distal subdomain of GalR dimer are important for repressosome formation. A further analysis of these and intragenic suppressors of looping-defective GalR mutants as well as gain-of-function mutants that permit repressosome assembly in the absence of HU show that GalR dimers contact each other in the repressosome in a partially stacked configuration.

Alanine↗

PubMed2002

Genetic instability favoring transversions associated with ErbB2-induced mammary tumorigenesis.

It has been argued that genetic instability is required to generate the myriad mutations that fuel tumor initiation and progression and, in fact, patients with heritable cancer susceptibility syndromes harbor defects in specific genes that normally maintain DNA integrity. However, the vast majority of human cancers arise sporadically, in the absence of deficiencies in known "mutator" genes. We used a cII-based mutation detection assay to show that the mean frequency of forward mutations in primary mammary adenocarcinomas arising in mouse mammary tumor virus-c-erbB2 transgenic mice harboring multiple copies of the lambda bacteriophage genome was significantly higher than in aged-matched, wild-type mammary tissue. Analysis of the cII mutational spectrum within the mammary tumor genomic DNA demonstrated a >6-fold elevation in transversion mutation frequency, resulting in a highly unusual inversion of the transition/transversion ratio characteristic of normal epithelium; frameshift mutation frequencies were unaltered. Arising oncogenic point mutations within the c-erbB2 transgene of such tumors were predominantly transversions as well. Data from this model system support the notion that elaboration of a mutator phenotype is a consequential event in breast cancer and suggest that a novel DNA replication/repair gene is a relatively early mutational target in c-erbB2-induced mammary tumorigenesis.

Adenocarcinoma↗

PubMed2002

Evolution of DNA binding motifs and operators.

A gene regulatory protein with helix-turn-helix (HTH) DNA-binding motif, GalS contains a functional operator within the DNA sequences encoding the HTH region (Nature 369 (1994) 314). We searched for operator-like sequences within the DNA sequences encoding the DNA binding motifs of other regulatory proteins. Five such proteins, DeoR, CytR, LRP, LuxR and PurR, were found to have actual operator or operator-like sequences in the DNA sequences encoding the DNA-binding motif. Except DeoR, all of them including GalS, are known to be auto-regulated. Auto-regulation in case of DeoR has not been investigated. Seven other proteins containing a HTH motif, do not have operator-like sequences in the DNA sequences encoding the HTH motif; none of them, except MerR, are known to be auto-regulated. The DNA binding proteins may have evolved from a common ancestor containing a DNA binding site within its gene segment that encodes the DNA-binding motif to facilitate auto-regulation. We have discussed current evidence for monophyletic or polyphyletic origin of such sequences.

Amino Acid Sequence↗

PubMed2002

Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium.

Colonization of the gastrointestinal tract with vancomycin-resistant Enterococcus faecium (VRE) has become endemic in many hospitals and nursing homes in the United States. Such colonization predisposes the individual to VRE bacteremia and/or endocarditis, and immunocompromised patients are at particular risk for these conditions. The emergence of antibiotic-resistant bacterial strains requires the exploration of alternative antibacterial therapies, which led our group to study the ability of bacterial viruses (bacteriophages, or phages) to rescue mice with VRE bacteremia. The phage strain used in this study has lytic activity against a wide range of clinical isolates of VRE. One of these VRE strains was used to induce bacteremia in mice by intraperitoneal (i.p.) injection of 10(9) CFU. The resulting bacteremia was fatal within 48 h. A single i.p. injection of 3 x 10(8) PFU of the phage strain, administered 45 min after the bacterial challenge, was sufficient to rescue 100% of the animals. Even when treatment was delayed to the point where all animals were moribund, approximately 50% of them were rescued by a single injection of this phage preparation. The ability of this phage to rescue bacteremic mice was demonstrated to be due to the functional capabilities of the phage and not to a nonspecific immune effect. The rescue of bacteremic mice could be effected only by phage strains able to grow in vitro on the bacterial host used to infect the animals, and when such strains are heat inactivated they lose their ability to rescue the infected mice.

Animals↗

PubMed2002

Bacteriophage SP6 is closely related to phages K1-5, K5, and K1E but encodes a tail protein very similar to that of the distantly related P22.

The lytic salmonella phage SP6 encodes a tail protein with a high degree of sequence similarity to the tail protein of the biologically unrelated lysogenic salmonella phage P22. The SP6 tail gene is flanked by an upstream region that contains a promoter and a downstream region that contains a putative Rho-independent transcription terminator, giving it a cassette or modular structure almost identical to the structure of the tail genes of coliphages K1E, K5, and K1-5. It now appears that SP6, K1-5, K5, and K1E are very closely related but have different tail fiber proteins, giving them different host specificities.

Amino Acid Sequence↗

PubMed2001

In vitro repression of the gal promoters by GalR and HU depends on the proper helical phasing of the two operators.

Repression of transcription initiation from the two gal promoters, P1 and P2, requires binding of GalR protein to two flanking operators, O(E) and O(I), binding of HU to a site, hbs, located between the two operators, and supercoiled DNA template. Previous experiments suggested that repression involves the interaction of two DNA-bound GalR proteins, which generates a 113-bp DNA loop encompassing the promoter region. Interaction between two DNA-bound proteins would be allowed if the binding sites on DNA are properly aligned. To test the idea that the observed repression of gal transcription in vitro is mediated by DNA looping, we investigated the effect of changing the relative angular orientation of O(E) and O(I) in the DNA helix. We found that repression is a periodic function of the distance between the two operator sites. Since repression recurred commensurate with DNA helical repeat, we conclude that the observed in vitro repression is mediated by DNA looping and the in vitro conditions reflect the in vivo situation.

Bacterial Proteins↗