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W S Reznikoff

Publications and source records attributed to W S Reznikoff.

At least 19 recordsLinked to original sources
PubMed1992

The lactose operon-controlling elements: a complex paradigm.

The lactose-controlling elements have been considered to be the simple paradigm of a cis-acting genetic regulatory system, containing a promoter whose activity is modulated by an operator and a catabolite gene activator protein (CAP)-binding site. The reality is considerably more complex. We now know that transcription is negatively regulated as a result of the repressor binding to three binding sites: the operator, a secondary repressor-binding site within the lacZ gene and a tertiary repressor-binding site upstream near lacI. In addition to the promoter, the lac-controlling elements contain five promoter-like elements. The physiological role, if any, of these promoter-like elements is not clear, although three of them can be activated by single base pair changes to give high levels of in vivo expression. Finally, the positive activator protein CAP has been found to bind to a secondary site which is coincident with the operator. No role has been identified for this secondary CAP-DNA complex.

Binding Sites↗
PubMed1992

Fis plays a role in Tn5 and IS50 transposition.

The Fis (factor for inversion stimulation) protein of Escherichia coli was found to influence the frequency of transposon Tn5 and insertion sequence IS50 transposition. Fis stimulated both Tn5 and IS50 transposition events and also inhibited IS50 transposition in Dam-bacteria. This influence was not due to regulation by Fis of the expression of the Tn5 transposition proteins. We localized, by DNase I footprinting, one Fis site overlapping the inside end of IS50 and give evidence to strongly suggest that when Fis binds to this site, IS50 transposition is inhibited. The Fis site at the inside end overlaps three Dam GATC sites, and Fis bound efficiently only to the unmethylated substrate. Using a mobility shift assay, we also identified another potential Fis site within IS50. Given the growth phase-dependent expression of Fis and its differential effect on Tn5 versus IS50 transposition in Dam-bacteria, we propose that the high levels of Fis present during exponential growth stimulate transposition events and might bias those events toward Tn5 and away from IS50 transposition.

Adenine↗
PubMed1992

Characterization of two hypertransposing Tn5 mutants.

Transposition of Tn5 in Escherichia coli is regulated by two transposon-encoded proteins: transposase (Tnp), promoting transposition preferentially in cis, and the trans-acting inhibitor (Inh). Two separate transposase mutants were isolated that replace glutamate with lysine at position 110 (EK110) and at position 345 (EK345). The EK transposase proteins increase the Tn5 transposition frequency 6- to 16-fold in cis and enhance the ability of transposase to act in trans. The purified mutant transposase proteins interact with transposon outside end DNA differently from the wild-type protein, resulting in the formation of a novel complex in gel retardation assays. During characterization of the transposase proteins in the absence of inhibitor, we found that wild-type transposase itself has a transposition-inhibiting function and that this inhibition is reduced for the mutant proteins. We present a model for the regulation of Tn5 transposition, which proposes the existence of two transposase species, one cis-activating and the other trans-inhibiting. The phenotype of the EK transposase mutants can be explained by a shift in the ratio of these two species.

Base Sequence↗
PubMed1991

Translation initiation of IS50R read-through transcripts.

IS50R (and Tn5) normally transposes at a low frequency, partly because cells containing this insertion sequence synthesize low levels of the transposase protein. Since the 5' end of the transposase gene is located next to the outer end of IS50R (and thus close to flanking host sequences), transposition into actively transcribed genes could result in the production of read-through transcripts that would encode the transposase. We have found that these read-through transcripts are made, but are translated poorly. We isolated mutations that increase translation initiation of transposase from read-through transcripts. Most of these mutations destabilize a potential RNA secondary structure in the ribosome binding site that could form in read-through transcripts, but not in normal transcripts. In vitro RNA secondary structure analysis has confirmed the predicted RNA secondary structure and the effects of mutations. We have shown that RNA secondary structure is the major factor limiting transposase expression from read-through transcripts.

Base Sequence↗
PubMed1991

Fnr mutants that activate gene expression in the presence of oxygen.

The regulatory protein Fnr is required for anaerobic expression of several anaerobic respiratory enzymes in Escherichia coli. To gain insight into how Fnr activity is regulated by oxygen, we have isolated Fnr mutants that increase expression of the nitrate reductase operon in the presence of oxygen (Fnr* mutants). Seven single-amino-acid substitutions that mapped within two regions of Fnr have been characterized. Two mutants mapped adjacent to two Cys residues in the N-terminal Cys cluster. Five Fnr* substitutions mapped to a region of Fnr that is similar to the cyclic AMP-binding domain of the catabolite activator protein (CAP). Within this group, four mutants were clustered in a region analogous to the CAP C helix, which is important in CAP dimer subunit interactions. Taken together, these data implicate regions in Fnr that may be important either in sensing oxygen deprivation or in the conformational change proposed to be necessary for Fnr activation under anaerobic conditions.

Aerobiosis↗
PubMed1991

Downstream deletion analysis of the lac promoter.

We have generated a series of deletions in the downstream region of the lac promoter. The promoter activities of these mutations were compared by measuring the levels of beta-galactosidase gene expression in vivo. Our results show that deletion of downstream lac promoter sequences changes the promoter strength only two- to threefold. The effects of these deletions on transcription initiation site location were studied through primer extension assay of in vivo mRNAs. We found that the transcription start sites are primarily chosen as an approximate distance from the -10 region of the lac promoter; however, starts are sometimes manifested at a GAATT(C) sequence, which is identical to the wild-type preferred start site. lac promoter P2 and a newly identified promoter, P3, are transcribed in vivo at low levels. Catabolite activator protein complexed with cyclic AMP represses P2 and P3 expression in vivo. The secondary catabolite activator protein binding site plays at most a modest role in catabolite repression in vivo.

Base Sequence↗
PubMed1991

Escherichia coli catabolite gene activator protein mutants defective in positive control of lac operon transcription.

We isolated three Escherichia coli catabolite gene activator protein mutants that are defective in the positive control of transcription initiation from the lac operon promoter region yet retain negative control of transcription from other promoters. One mutant has a substitution of valine for glutamate at residue 72, which lies in the cyclic AMP binding domain and contacts cyclic AMP. The other two mutants have substitutions of asparagine and cysteine for glycine 162, which lies in a surface-exposed turn of the DNA-binding domain. Surprisingly, although all three mutants can repress the lacP2/P3 promoters through the catabolite gene activator protein target site of lac, none displays strong dominance over the ability of wild-type catabolite gene activator protein to stimulate the lacP1 promoter.

Base Sequence↗
PubMed1991

Anaerobically expressed Escherichia coli genes identified by operon fusion techniques.

Genes that are expressed under anaerobic conditions were identified by operon fusion techniques with a hybrid bacteriophage of lambda and Mu, lambda placMu53, which creates transcriptional fusions to lacZY. Cells were screened for anaerobic expression on XG medium. Nine strains were selected, and the insertion point of the hybrid phage in each strain was mapped on the Escherichia coli chromosome linkage map. The anaerobic and aerobic expression levels of these genes were measured by beta-galactosidase assays in different medium conditions and in the presence of three regulatory mutations (fnr, narL, and rpoN). The anaerobically expressed genes (aeg) located at minute 99 (aeg-99) and 75 (aeg-75) appeared to be partially regulated by fnr, and aeg-93 is tightly regulated by fnr. aeg-60 requires a functional rpoN gene for its anaerobic expression. aeg-46.5 is repressed by narL. aeg-65A and aeg-65C are partially controlled by fnr but only in media containing nitrate or fumarate. aeg-47.5 and aeg-48.5 were found to be anaerobically induced only in rich media. The effects of a narL mutation on aeg-46.5 expression were observed in all medium conditions regardless of the presence or absence of nitrate. This suggests that narL has a regulatory function in the absence of exogenously added nitrate.

Anaerobiosis↗
PubMed1991

Induction of the SOS response in Escherichia coli inhibits Tn5 and IS50 transposition.

In response to DNA damage or the inhibition of normal DNA replication in Escherichia coli, a set of some 20 unlinked operons is induced through the RecA-mediated cleavage of the LexA repressor. We examined the effect of this SOS response on the transposition of Tn5 and determined that the frequency of transposition is reduced 5- to 10-fold in cells that constitutively express SOS functions, e.g., lexA(Def) strains. Furthermore, this inhibition is independent of recA function, is fully reversed by a wild-type copy of lexA, and is not caused by an alteration in the levels of the Tn5 transposase or inhibitor proteins. We isolated insertion mutations in a lexA(Def) background that reverse this transposition defect; all of these mapped to a new locus near 23 min on the E. coli chromosome.

Alleles↗
PubMed1990

In vitro secondary structure analysis of mRNA from lacZ translation initiation mutants.

mRNA secondary structure can be an important determinant of the efficiency of translation initiation. To study the effect of secondary structure on translation initiation, in vitro secondary structure analysis was performed on 32 lacZ RNA transcripts that differ in their in vivo translation initiation efficiencies because of mutations. We have shown that well-translated RNA has a relatively unstructured translation initiation region in vitro. In contrast, the translation initiation region of many of the poorly translated RNA transcripts is involved in a stem-loop structure. Mutations that decrease the in vitro stability of the stem-loop increase the frequency of translation initiation. The sequences responsible for forming this stem-loop structure were localized to a small region of RNA. The results confirm some of the previous predictions of the RNA secondary structure of the mutant RNAs based on computer modeling, but they disagree with some of the predicted long-range interactions.

Base Composition↗
PubMed1990

Integration host factor plays a role in IS50 and Tn5 transposition.

In Escherichia coli, the frequencies of IS50 and Tn5 transposition are greater in Dam- cells than in isogenic Dam+ cells. IS50 transposition is increased approximately 1,000-fold and Tn5 transposition frequencies are increased about 5- to 10-fold in the absence of Dam methylation. However, in cells that are deficient for both integration host factor (IHF) and Dam methylase, the transposition frequencies of IS50 and Tn5 approximate those found in wild-type cells. The absence of IHF alone has no effect on either IS50 or Tn5 transposition. These results suggest that IHF is required for the increased transposition frequencies of IS50 and Tn5 that are observed in Dam- cells. It is also shown that the level of expression of IS50-encoded proteins, P1 and P2, required for IS50 and Tn5 transposition and its regulation does not decrease in IHF- or in IHF- Dam- cells. This result suggests that the effects of IHF on IS50 and Tn5 transposition are not at the level of IS50 gene expression. Finally, IHF is demonstrated to significantly retard the electrophoretic mobility of a 289-base-pair segment of IS50 DNA that contains a putative IHF protein-binding site. The physiological role of this IHF binding site remains to be determined.

Bacterial Proteins↗
PubMed1989

Pseudorevertants of a lac promoter mutation reveal overlapping nascent promoters.

Four pseudorevertants of a -10 region lacP mutation were isolated. Three of these mutations were found to activate nascent promoters. These mutations were: a -2 G/C----A/T change (-2A) promoting transcription at position +11, a +1 A/T----T/A change (+1T) promoting transcription initiation at position +13, and a +10 C/G----A/T change (+10A) promoting transcription initiation at a complex series of positions. The fourth mutation [a -12 T/A----A/T change (-12A)] promotes transcription initiation at -1. The promoters activated by mutations -12A, -2A and +1T resembled the canonical sigma 70 promoter sequences. The +10A promoter activity is also dependent upon the sigma 70 holoenzyme but can not be readily assigned to a specific promoter sequence.

Escherichia coli↗
PubMed1989

Galactose-1-phosphate uridylyltransferase: identification of histidine-164 and histidine-166 as critical residues by site-directed mutagenesis.

Galactose-1-phosphate uridylyltransferase catalyzes the interconversion of UDP-glucose and galactose-1-P with UDP-galactose and glucose-1-P by a double-displacement mechanism involving the compulsory formation of a uridylyl enzyme intermediate. The uridylyl group is covalently bonded to the N3 position of a histidine residue in the uridylyl enzyme. The galT gene of Escherichia coli, which codes for the uridylyltransferase and is contained in a plasmid for transformation of E. coli, has been sequenced, and the positions of the 15 histidine residues have been determined from the deduced amino acid sequence of this protein. Fifteen mutant genes, in each of which one of the 15 histidine codons has been changed to an asparagine codon, have been generated and used to transform the E. coli strain JM101. When extracts of the transformants were assayed for uridylyltransferase, 13 exhibited high levels of activity. Two of the extracts containing mutant uridylyltransferase exhibited less than control levels of activity. These mutant proteins, H164N and H166N, were overexpressed, isolated, and tested for their ability to form the compulsory uridylyl enzyme intermediate. Neither the H164N nor the H166N mutant proteins could form the intermediate. Thus, both His-164 and His-166 are critical for activity, and their proximity suggests that both are in the active site. One is the essential nucleophilic catalyst to which the uridylyl group is bonded in the intermediate, and the other serves an equally important, as yet unknown, function. The active-site sequence His(164)-Pro-His(166) is conserved in this enzyme from E. coli, humans, Saccharomyces, and Streptomyces.

Asparagine↗
PubMed1989

Orientation of IS50 transposase gene and IS50 transposition.

Reversal of transposase gene orientation with respect to the nonidentical ends of IS50 strongly decreased IS50 transposition in both Dam- and Dam+ hosts. In either orientation, IS50 transposase expression was unaffected. These effects were independent of the surrounding DNA context. This shows that the efficiency of IS50 transposition is dependent on transposase gene orientation. The transposition frequencies of transposons utilizing inverted IS50 inside ends (IE), IE-IE transposons, were lower than either outside end (OE)-IE or OE-OE transposons.

Base Sequence↗
PubMed1988

Use of a Tn5 derivative that creates lacZ translational fusions to obtain a transposition mutant.

We constructed a derivative of Tn5, Tn5 ORFlac, that is capable of creating lacZ translational fusions upon transposition. Lac- strains carrying this construct formed red papillae when plated on MacConkey-lactose media. Lac+ cells isolated from independent papillae expressed distinct beta-galactosidase fusion proteins, suggesting that the Lac+ phenotype resulted from transposition. In support of this, analysis of plasmids carrying Tn5 ORFlac prepared from these cells indicated that the Lac+ phenotypes arose as a result of intermolecular rearrangements. Furthermore, a derivative of Tn5 ORFlac that contains an ochre mutation in the transposase gene formed papillae only in a supB strain. Tn5 ORFlac is useful for obtaining mutants that affect Tn5 transposition and for creating lacZ fusions. We used the papillation phenotype to isolate a spontaneous revertant of IS50L that promotes transposition at a 3.6-fold higher rate than IS50R. The mutation altered the amino acid sequence of both transposase and inhibitor.

Base Sequence↗
PubMed1988

Effect of dam methylation on Tn5 transposition.

The effect of dam methylation on Tn5 transposition was investigated by analyses of mutations in the host (Escherichia coli) and the element. Wild-type elements transposed at a higher frequency and showed higher levels of transposase expression in a dam-host. Mutations were made in the promoter region of the transcript that codes for the transposase. Transposition and transposase levels from these mutants were independent of the host methylation system. Measurements of the amount of RNA support the hypothesis that dam methylation exerts its effect on Tn5 transposition by modulating the frequency of transcriptional initiation of the transposase gene. Since Tn5 transposition increases when the transposase levels increase, at normal concentrations the amount of transposase is a rate-limiting factor that determines the transposition frequency of Tn5. Transposition of IS50, one of the insertion sequences that constitutes Tn5, is also sensitive to dam methylation by a second mechanism in addition to that of modulating transcriptional initiation. dam methylation, either directly or indirectly, inhibits the usage of IS50 sequences by the transposase. Thus, dam methylation can affect both the expression of the transposase and the DNA substrate upon which it acts.

DNA Transposable Elements↗