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S Adhya

Publications and source records attributed to S Adhya.

At least 109 records · Page 6Linked to original sources
PubMed1984

Demonstration of two operator elements in gal: in vitro repressor binding studies.

Genetic and DNA base sequence analyses of cis-dominant mutations that derepress the gal operon of Escherichia coli suggested the existence of two operator loci needed for gal repression. One (OE) is located immediately upstream to the two overlapping gal promoters and the other (OI) is inside the first structural gene. We have investigated the ability of wild-type and mutant OE and OI DNA sequences to bind to gal repressor. The repressor has been purified from cells containing a multicopy plasmid in which the repressor gene is brought under the control of phage lambda PL promoter. The DNA-repressor interactions are detected by the change in electrophoretic mobility of labeled DNA that accompanies its complex formation with repressor protein. The purified repressor shows concentration-dependent binding to both O+E and O+I but not to OEc and OIc sequences. These results authenticate the proposed operator role of the two homologous gal DNA control elements and thereby establish that the negative control of the gal operon requires repressor binding at both OE and OI, which are separated by greater than 90 base pairs.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

A control element within a structural gene: the gal operon of Escherichia coli.

The gal operon of Escherichia coli is transcribed from two overlapping promoters, PG1 and PG2. Cyclic AMP and its receptor protein (CRP) modulate the two promoters in opposite directions by binding to a single cat locus. Both the promoters are negatively regulated by a single repressor, the product of the galR gene. An operator site, defined by several mutations, has previously been located upstream from the cat locus. We have isolated and characterized a new set of cis-dominant constitutive mutations of the gal operon and determined their locations by DNA sequencing. From these studies, we propose the existence of a second functional gal operator element at an extraordinary site--within galE, the first structural gene. Both the operators, OE (exterior) and OI (interior), are involved in the repression of PG1 and PG2. This would be the first example of the presence of a functional operator element within a structural protein-coding region.

Base Sequence↗
PubMed1983

Cyclic AMP-dependent constitutive expression of gal operon: use of repressor titration to isolate operator mutations.

When the gal operator region is present in a multicopy plasmid it binds to all ("titrates") the gal repressor and "induces" the chromosomal gal operon. To make operator mutations (Oa) with reduced affinity toward the repressor, plasmid DNA was irradiated with UV light and mutant derivatives were isolated that were unable to release the chromosomal gal genes from repression. Then with such an Oa plasmid operator revertants were isolated that had reacquired the ability to release repression. Both sets of mutations have been localized by DNA sequence analysis. When the Oa mutations were transferred from the plasmid to the chromosome by recombination these mutant operators were found to make gal expression constitutive (independent of repressor) but still dependent on cAMP, whereas the previously reported gal operator mutants (Oc) are constitutive both in the presence and in the absence of cAMP. The titration method of isolating mutants enables the isolation of strains with operator mutations that also affect normal promoter activity, and it provides an easy way to isolate revertants of operator mutations.

Base Sequence↗
PubMed1983

The pleiotropic ts15 mutation of E. coli is an IS1 insertion in the rho structural gene.

Rho protein regulates transcription termination in E. coli. Some of the temperature-sensitive mutants defective in Rho protein, e.g., ts15, show remarkable pleiotropic phenotypes. The ts mutations map between the ilv and cya loci on the E. coli chromosome. We have cloned the gene that restores the wild-type phenotypes of these mutants. Genetic and biochemical characterizations have shown that the cloned DNA segment carries the structural gene for the Rho polypeptide. Analysis of the rhots15 mutation has revealed the presence of an IS1 insertion in the carboxy terminal segment of the rho cistron, thereby truncating the 52-kilodalton (kd) Rho polypeptide to a 50-kd size and also making it thermolabile. This provides an example of how an IS1 insertion mutation can cause a TS phenotype. We have also shown that the multiple phenotypes of the mutant cell, including the temperature sensitivity, are caused by a single mutation (rhots15::IS1) in the rho structural gene. How a rho structural gene mutation may cause such pleiotropy is discussed.

Bacterial Proteins↗
PubMed1983

Regulation of the pR operon of bacteriophage lambda.

The E. coli lambda lysogen, OR1263, carries the fusion pR-cro-tR1-IS2-gal. The gal promoter is deleted and gal expression from pR, in the absence of the lambda antitermination factor N, is blocked by the efficient transcription terminator in IS2. Selection for Gal+ yields strains deleted for the IS2 terminator and various portions of the lambda chromosome. Analysis of these deletions reveals the following: (a) The lambda tR1 terminator is about 50% efficient. (b) In two deletions sequenced, DNA loss occurred as a result of homologous recombination between a 2- or a 4-base pair repeat. (c) By measuring the ability of lambda N product to suppress the polarity of a gal ochre mutation, we demonstrate that the N utilization site in the lambda pR operon lies between tR1 and cro. (d) The level of Cro repressor synthesized by a single copy prophage is sufficient to repress the cI maintenance promoter, prm, but is inadequate to inhibit pR.

Bacteriophage lambda↗
PubMed1982

Promoter occlusion: transcription through a promoter may inhibit its activity.

Induction of prophage lambda inhibits the expression of the gal operon from its cognate promoters. The effect is observed only in cis, and is due to frequent transcription of the gal promoter region by RNA polymerase molecules initiating upstream at the prophage PL promoter. The frequency of transcription initiation at PL is some 30 times greater than that at the gal promoter, Pg1. PL is one of the strongest procaryotic promoters. This "promoter occlusion" is essentially complete when the distance between gal and PL is small (less than or equal to 10 kb); and when PL is fully active (that is, in the absence of the cl or cro repressors). We discuss the possibility that promoter occlusion at two lambda promoters, Pint and PR', might play a role in the sequential expression of viral functions.

Bacteriophage lambda↗
PubMed1982

Cloning of the RHO gene of Escherichia coli.

The map position of the rho gene on E coil chromosome, as determined by phage P1 generalized transduction, was determined. The gene order is ilv-rho-cya. By Hind III endonuclease shotgun technique, the rho+ gene was cloned into a lambda-Charon 25 vector. The sizing of the restriction endonuclease generated DNA fragments by agarose gel electrophoresis, the heteroduplex analysis of cloned DNA molecules by electron microscope and the demonstration of the synthesis of rho protein by the cloned DNA shows that a functional piece of DNA, containing the entire rho gene of E coli has been cloned.

Bacteriophage lambda↗
PubMed1981

Evidence for two functional gal promoters in intact Escherichia coli cells.

We have used an S1 mapping assay to demonstrate that the mRNA transcripts of the Escherichia coli galactose operon found in intact E. coli cells with a defect in adenylate cyclase or the cyclic AMP receptor protein contain at their 5' end about five nucleotides more than the gal mRNA molecules made in wild type cells. The same difference between gal RNA synthesized in vitro in the absence of cyclic AMP or cyclic AMP receptor protein and gal RNA made in the presence of these factors is detected by this assay. Our results strongly suggest that the same two overlapping promoters, which we previously identified by in vitro transcription of gal DNA fragments, also control the expression of the galactose operon in intact cells. The intracellular levels of cyclic AMP determine which promoter is utilized.

DNA, Bacterial↗
PubMed1981

Location, function, and nucleotide sequence of a promoter for bacteriophage T3 RNA polymerase.

The major promoters for bacteriophage T3 RNA polymerase on the T3 genome have been mapped by DNA.RNA filter hybridization. One promoter is located in a 300-base-pair Hpa I restriction fragment near the genetic "left" end of T3 DNA. The sequence in the vicinity of the major initiation site of transcription in this region has been determined. A part of the (-)strand sequence is 5' T-A-T-T-T-A-C-C-C-T-C-A-C-T-A-A-A-G-+1 G-G-A-A-U 3'. Comparison of this sequence with the prototype 23-base-pair promoter sequence for bacteriophage T7 RNA polymerase shows a striking pattern of homology and divergence. Between positions -9 and +4, the sequences are virtually identical, whereas between positions -17 and -10, the sequences are quite different. It is postulated that these sequence subsets may perform different functions in transcription initiation by the phage RNA polymerases.

Base Sequence↗
PubMed1981

Guanylate cyclase activity in Escherichia coli mutants defective in adenylate cyclase.

Guanylate cyclase, which catalyzes the synthesis of guanosine 3',5'-monophosphate, has been assayed in several strains of Escherichia coli. They include wild-type cells and mutants defective in adenylate cyclase, which is responsible for the synthesis of adenosine 3',5'-phosphate. Our results demonstrate that adenylate cyclase and guanylate cyclase are two different enzymes in E. coli and suggest that the gene that encodes adenylate cyclase also plays a regulatory role in the synthesis of guanylate cyclase.

Adenylyl Cyclases↗
PubMed1980

Construction of a restriction map of bacteriophage T3 DNA.

A restriction endonuclease cleavage map of bacteriophage T3 DNA has been constructed. The enzymes used and, within parentheses, the number of their cleavage sites on T3 DNA are: HindIII (1), XbaI (1), BglII (1), KpnI (2), MboI (9), and HpaI (17). The size and the relative location of each fragment have been established, defining an accurate physical map of T3 DNA.

Chromosome Mapping↗
PubMed1980

L factor that is required for beta-galactosidase synthesis is the nusA gene product involved in transcription termination.

The DNA-dependent in vitro synthesis of Escherichia coli beta-galactosidase requires the presence of a soluble protein referred to as L factor [Kung, H., Spears, C. & Weissbach, H. (1975) J. Biol. Chem. 250, 1556-1562]. In the present study, comparison of physical, immunological, and biological properties shows that L factor is the product of the E. coli nusA gene. The nusA gene product is known to interact with bacteriophage lambda N gene protein and to prevent premature termination of transcription from the early lambda promoters. Our results suggest that premature transcription termination in the lac operon of E. coli may also be overcome by the nusA protein.

Bacterial Proteins↗
PubMed1979

Termination of transcription by Escherichia coli RNA polymerase: influence of secondary structure of RNA transcripts on rho-independent and rho-dependent termination.

The effect of RNA secondary structure on rho-independent and rho-dependent termination of transcription of T3 DNA by Escherichia coli RNA polymerase has been studied by incorporating, into nascent transcripts, base analogs that lead to altered base-pairing properties. A guanine --> hypoxanthine substitution, with attendant weakening of secondary structure, abolished the rho-independent termination at 20% of the genome; in contrast, replacement of cytosine with 5-bromocytosine, which forms stronger pairs with guanine, enhanced termination at this site. rho-Independent termination was not altered by replacing uracil with 5-bromouracil. There are two major rho-dependent termination sites on the T3 DNA-at 8 and 15%. The termination activity of rho in this system also depended on RNA secondary structure. The incorporation of 5-bromouracil instead of uracil into RNA did not alter the site specificity of rho action but rho was rendered inactive when cytosine was replaced by 5-bromocytosine. In contrast, replacement of GTP with ITP in the reaction increased rho-dependent inhibition of RNA synthesis, caused production of heterogeneous-sized transcripts, and stimulated rho-mediated ATP hydrolysis. The rho-associated ATPase activity, in the presence of isolated T3 RNA, was also stimulated by inosine substitution. Furthermore, the temperature-sensitive rho isolated from rho 15 mutant of E. coli, which does not terminate transcription in the presence of the common rNTPs, was active when GTP was replaced with ITP. These results suggest that strongly paired G.C-rich regions in RNA stem-loop structures or RNA.DNA hybrids are essential for rho-independent termination, whereas rho-dependent termination requires weakly paired cytosine residues for its action.

Adenosine Triphosphatases↗