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P L deHaseth

Publications and source records attributed to P L deHaseth.

33 records · Page 2Linked to original sources
PubMed1989

Promoter recognition by Escherichia coli RNA polymerase. Role of the spacer DNA in functional complex formation.

The available evidence suggests that during the process of formation of a functional or "open" complex at a promoter, Escherichia coli RNA polymerase transiently realigns the two contacted regions of the promoter, thus stressing the intervening spacer DNA. We tested the possibility that this process plays an active role in the formation of an open complex. Two series of promoters were examined: one with spacer DNAs of 15 to 19 base-pairs and a derivative for which the promoters additionally contained a one-base gap in the spacer, so as to relieve any stress imposed on the DNA. Consistent with an active role for the stressed DNA in driving open complex formation, we have found that for promoters with a 17-base-pair spacer, the presence of a gap leads to a delay in the formation of an open complex, at a step subsequent to the initial binding of RNA polymerase to the promoter. The results with the other gapped promoters rule out direct binding of RNA polymerase to the region of the gap and indicate an increased flexibility in the gapped DNA. As not all observations with the spacer length series of gapped and ungapped promoters can be interpreted in terms of an active role of the spacer DNA without additional assumptions, such a role must still be considered tentative.

Base Sequence↗
PubMed1989

Saturation mutagenesis of an Escherichia coli rRNA promoter and initial characterization of promoter variants.

Using oligonucleotide synthesis techniques, we generated Escherichia coli rrnB P1 (rrnB1p according to the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoter fragments containing single base substitutions, insertions, deletions, and multiple mutations, covering the whole length of the promoter including the upstream activation sequence (UAS). The activities of 112 mutant promoters were assayed as operon fusions to lacZ in lambda lysogens. The activities of most mutants with changes in the core promoter recognition region (i.e., substitutions, insertions, or deletions in the region of the promoter spanning the -10 and -35 E. coli consensus hexamers) correlated with changes toward or away from the consensus in the hexamer sequences or in the spacing between them. However, changes at some positions in the core promoter region not normally associated with transcriptional activity in other systems also had significant effects on rrnB P1. Since rRNA promoter activity varies with cellular growth rate, changes in activity can be the result of changes in promoter strength or of alterations in the regulation of the promoter. The accompanying paper (R. R. Dickson, T. Gaal, H. A. deBoer, P. L. deHaseth, and R. L. Gourse, J. Bacteriol. 171:4862-4870, 1989) distinguishes between these two alternatives. Several mutations in the UAS resulted in two- to fivefold reductions in activity. However, two mutants with changes just upstream of the -35 hexamer in constructs containing the UAS had activities 20- to 100-fold lower than the wild-type level. This collection of mutant rRNA promoters should serve as an important resource in the characterization of the mechanisms responsible for upstream activation and growth rate-dependent regulation of rRNA transcription.

Bacteriophage lambda↗
PubMed1989

Identification of promoter mutants defective in growth-rate-dependent regulation of rRNA transcription in Escherichia coli.

We measured the activities of 50 operon fusions from a collection of mutant and wild-type rrnB P1 (rrnB1p in the nomenclature of B. J. Bachmann and K. B. Low [Microbiol. Rev. 44:1-56, 1980]) promoters under different nutritional conditions in order to analyze the DNA sequence determinants of growth rate-dependent regulation of rRNA transcription in Escherichia coli. Mutants which deviated from the wild-type -10 or -35 hexamers or from the wild-type 16-base-pair spacer length between the hexamers were unregulated, regardless of whether the mutations brought the promoters closer to the E. coli promoter consensus sequence and increased activity or whether the changes took the promoters further away from the consensus and reduced activity. These data suggest that rRNA promoters have evolved to maintain their regulatory abilities rather than to maximize promoter strength. Some double substitutions outside the consensus hexamers were almost completely unregulated, while single substitutions at several positions outside the -10 and -35 consensus hexamers exerted smaller but significant effects on regulation. These studies suggest roles for specific promoter sequences and/or structures in interactions with regulatory molecules and suggest experimental tests for models of rRNA regulation.

Escherichia coli↗
PubMed1988

Promoter recognition by Escherichia coli RNA polymerase. Influence of DNA structure in the spacer separating the -10 and -35 regions.

Escherichia coli RNA polymerase contacts promoter DNA at two regions (the -10 and -35 regions) which are separated by a segment of spacer DNA. Previously we showed that base substitutions in the spacer DNA can affect promoter strength both in vitro and in vivo; these results were interpreted to reflect altered structural properties of the substituted DNAs. Here we provide experimental support for this interpretation. The pattern of cleavage of the promoters with Neurospora crassa endonuclease and the reactivity of their guanine residues with dimethyl sulfate (DMS) suggest that the structures of the spacer DNAs in the promoters with altered transcriptional activities are distinct. In addition, the binding of RNA polymerase to the latter promoters induces characteristic enhancements in the extent to which specific guanine residues in the spacer DNAs react with DMS. We propose that for these promoters the substitutions in the spacer DNAs have affected the relative orientation of the -10 and -35 regions. The observed differences in promoter activity then would reflect the requirement for realignment of these regions during the process of open complex formation; we postulate that two such realignments occur.

Autoradiography↗
PubMed1987

Promoter recognition by Escherichia coli RNA polymerase: effects of base substitutions in the -10 and -35 regions.

We have constructed the PRM promoter of phage lambda and eight variants, which represents intermediates in the conversion of this promoter to one that has complete homology to the consensus sequences in the -10 and -35 regions. The in vivo activity of these promoters was determined from the beta-galactosidase or galactokinase activities in cells harboring plasmids, in which the cloned promoters were driving the expression of these genes. Additionally, the kinetics of the interaction of Escherichia coli RNA polymerase with the same series of promoters was measured as a function of RNA polymerase concentration. This allowed the overall rate of functional or open complex formation to be dissected into the equilibrium constant for binding of the polymerase to form a closed promoter complex and the rate of subsequent isomerization to yield the open complex. The following conclusions can be drawn from the data presented: (1) The consensus sequence is optimal for promoter function both in vivo and in vitro. (2) Alterations of the -10 and -35 regions have similar effects on the kinetics of RNA polymerase binding in vitro; with one exception, the same holds for promoter activity in vivo. (3) The in vitro rate of RNA polymerase binding to a promoter is solely determined by the number of positions at which its -10 and -35 regions match the consensus promoter sequence. The functional importance of a match does not appear to be determined by the sequence conservation at the particular position. (4) The extent to which a particular base change affects the kinetic parameters depends on the sequence of the promoter into which it is introduced.

Bacteriophage lambda↗
PubMed1987

Specific activation of transcription initiation by the sequence-specific DNA-binding agents distamycin A and netropsin.

A series of promoters with nine base-pair substitutions in the spacer DNA separating the -10 and -35 regions was used to demonstrate that Escherichia coli RNA polymerase is sensitive to events affecting the spacer DNA--a region not directly contacted by the enzyme. The drugs distamycin A and netropsin specifically enhanced the rate of functional complex formation at a promoter bearing a substitution of nonalternating A-T base pairs. The effect is exerted at an early step in the RNA polymerase-promoter interaction. We hypothesize that a drug-induced structural alteration in the spacer DNA occurs, similar to that normally resulting from RNA polymerase binding. These findings are relevant to an understanding of potential mechanisms of transcription activation.

Base Sequence↗
PubMed1987

DNA functional groups required for formation of open complexes between Escherichia coli RNA polymerase and the lambda PR promoter. Identification via base analog substitutions.

Synthetic 75-base pair promoters bearing base changes and/or base analog substitutions at selected positions were constructed. Using both abortive initiation and run-off transcription assays, the interaction of these altered promoters with Escherichia coli RNA polymerase was studied in order to determine the involvement of DNA functional groups in promoter recognition. Two adjacent thymines in the -35 region were identified whose 5-methyl groups play a crucial role. Additionally, the combined results from several substitution experiments showed that functional groups in the major groove of the strongly conserved T-A base pair at the -7 position are probable sites of direct interaction with RNA polymerase.

Bacteriophage lambda↗
PubMed1986

Promoter recognition by Escherichia coli RNA polymerase. Effects of substitutions in the spacer DNA separating the -10 and -35 regions.

A family of variants of the PRM promoter of lambda phage was constructed, bearing nine base pair substitutions in a stretch of the spacer DNA separating the contacted -10 and -35 regions. The substituted sequences were chosen for their potential to adopt structures different from those of average B-form DNA and thus to affect the interaction of RNA polymerase with the two contacted regions. Characterization of the promoters in vitro and in vivo provides additional support for the lack of specific contacts in the substituted spacer region and shows that a small change in the relative rotational orientation of the -10 and -35 regions is inconsequential to promoter function. However, a 2-3-fold reduction in promoter activity is observed with promoters bearing substitutions of nonalternating dG-dC base pairs in either orientation. This corroborates other studies indicating the anomalous behavior of such sequences and suggests that the structure of the spacer DNA can modulate promoter recognition.

Bacteriophage lambda↗
PubMed1983

Chemical synthesis and biochemical reactivity of bacteriophage lambda PR promoter.

By a combination of chemical and enzymatic methods, a 75 base pair DNA duplex containing the sequence of the lambda PR promoter including the OR1 and OR2 cI repressor binding sites was synthesized. The solid support phosphite triester procedure (Caruthers, M. H. et al., Cold Spring Harbor Symposia on Quantitative Biology XLVII, in press) was used for the synthesis of oligonucleotides comprising the sequence. We report here an adaptation of the method of DNA synthesis in test tubes. Assembly of the oligonucleotides involved the use of T4 polynucleotide kinase and T4 DNA ligase. We show that the synthetic DNA is recognized by RNA polymerase and cI repressor in a manner identical to the same control region contained on a restriction fragment isolated from bacteriophage lambda DNA. Our synthetic approach using chemically synthesized promoter variants is thus suitable for studies probing the function of promoters.

Bacteriophage lambda↗
PubMed1978

Nonspecific interactions of Escherichia coli RNA polymerase with native and denatured DNA: differences in the binding behavior of core and holoenzyme.

We have investigated the nonspecific interactions of Escherichia coli RNA polymerase core and holoenzyme with double-stranded (ds) and single-stranded (ss) DNA. Binding constants for these interactions as functions of such solution variables as monovalent and/or divalent cation concentration, temperature, or pH were determined by the method of deHaseth et a. [deHaseth, P.L., Gross, C.A., Burgess, R.R. and Record, M.T. (1977), Biochemistry 16, 4777--4783] from analysis of the elution of the proteins from small columns containing immobilized DNA. This technique, although as yet empirical, has been demonstrated to yield accurate binding constants fot the nonspecific interation of lac repressor with ds DNA. We find that observed binding constants (Kobsd) are extraordinarily sensitive functions of the monovalent cation concentration for the interactions of both core and holoenzyme with ds DNA. In the absence of divalent cations, the derivatives --(d log Kobsd/d log [Na+]) are 11 +/- 2 for the holo--ds DNA interaction and 21 +/- 3 for the core--ds DNA interaction. Consequently, approximately 11 and 21 low-molecular-weight ions are released, iin the thermodynamic sense, in the formation of the holo--ds and core--ds complexes, respectively (Record, M.T., Jr., Lohman, T.M., and deHaseth, P.L. (1976), J. Mol. Biol. 107, 145--158; Record, M.T., Jr., Anderson, C.F., and Lohman, T.M. (1978), Q. Rev. Biophys., in press). Ion release is a thermodynamic driving force for these nonspecific interactions and causes the stability of the complexes to increase very substantially with a reduction in monovalent ion concnetration. Possible molecular models which account for the different salt sensitivities of the holo--ds and core--ds complexes are discussed. Effects of the competitive ligand Mg2+ on these interactions are also examined. Substantial ion release (approximately 18 monovalent ions) also accompanies the interaction of either holo or core polymerase with ss DNA. Over the range of ion concentrations investigated the holo--ss interaction is substantially stronger than the core--ss interaction; furthermore, we conclude that the interactions of polymerase with ss DNA are, in general, stronger than the nonspecific interations of the enzyme with ds DNA. It is likely that the nonspecific interactions of RNA polymerase with DNA have physiological relevance. Not only is it plausible to assume that the same regions of the protein are involved in both specific and nonspecific interactions, but in addition nonspecific interactions of RNA polymerase and DNA may play role in determining the availability of this protein, in both the thermodynamic and the kinetic sense, for promoter binding and RNA chain initiation [von Hippel. P.H., Revzin, A., Gross, C.A., and Wang, A.C. (1974), Proc. Natl. Acad. Sci U.S.A. 71, 4808--4812]. Consequently, the strong dependences of the nonspecific interactions of RNA polymerase on ionic conditions suggest the possibility of a modulating role of ion concentrations in the control of transcription.

DNA↗
PubMed1977

Measurement of binding constants for protein-DNA interactions by DNA-cellulose chromatography.

We describe the application of DNA-cellulose chromatography to the determination of binding constants for the nonspecific interaction between proteins and DNA. The method involves loading a small DNA-cellulose column to a low binding density and subsequently eluting the protein with buffer at a constant salt concentration. The elution is conveniently followed by monitoring the protein fluorescence of the eluate. From the shape of the elution profile, the binding constant for the interaction can be calculated. Employing columns containing double-stranded calf thymus DNA-cellulose, we have measured binding constants in the range of 10(4) to 10(6) M-1. Extension of this range is possible by varying the amount of DNA on the column. For lac repressor, agreement between our measurements and those of Revzin, A., and von Hippel, P.H. [(1977) Biochemistry 16 (second of five papers in a series in this issue)], who employ an absolute boundary sedimentation method, is good. The column method should be useful for the rapid screening of the effect of a large number of variables on protein-DNA binding constants.

Bacterial Proteins↗
PubMed1977

Nonspecific interaction of lac repressor with DNA: an association reaction driven by counterion release.

We have investigated the nonspecific interaction of lac repressor protein with DNA by a quantitative application of DNA-cellulose chromatography (deHaseth, P.L., et al. (1977), Biochemistry 16 (third of five papers in a series in this issue)). The observed association constant for the interaction, KRD obsd, is a sensitive function of ion concentrations and pH. Application of binding theory to interpret these effects gives the results that 11 +/- 2 monovalent ions are released in the interaction and two groups on repressor must be protonated for repressor to bind to DNA. We argue that much of the ion release results from the displacement of cations from the DNA, and estimate on this basis that 12 +/- 2 phosphates are involved in ionic interactions with the protein. Ion release drives the protonation reaction and the overall repressor-DNA interaction. The major role of low molecular weight ions in the repressor-DNA interaction suggests that ion concentration changes must be considered in discussing mechanisms of control of gene expression.

Bacterial Proteins↗
PubMed1977

Interpretation of monovalent and divalent cation effects on the lac repressor-operator interaction.

We have investigated the effects of mixed Na+: Mg2+ ionic solutions on the stability of the nonspecific lac repressor-DNA complex. The effects of Mg2+ are simply interpreted in terms of its role as a competitor (with repressor) for DNA sites. From these studies, the binding constant of the Mg-DNA complex can be determined as a function of the concentration of Na+. We have used this information to interpret the data of Riggs and collaborators (Riggs, A.D., et al. (1970), J. Mol. Biol. 48, 67-83; 53, 401-417) on the ion dependence of the repressor-operator interaction. We find that there are approximately 70% as many ionic interactions in the repressor-operator complex as in the nonspecific complex. Our best estimate is that 8 +/- 1 ion pairs are formed. We calculate that the release of counterions in the formation of the specific complex contributes approximately 40% of the favorable free energy change in the association reaction under in vivo ionic conditions. Implications of these findings for the control of the lac operon and for the molecular relationship between the specific and nonspecific complexes are considered.

Bacterial Proteins↗