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C Reiss

Publications and source records attributed to C Reiss.

At least 19 recordsLinked to original sources
PubMed1995

Kinetic study in vitro of Escherichia coli promoter closure during transcription initiation.

The rate of closure of two Escherichia coli promoters borne by plasmid pBR322, following transcription initiation from the open complex, was probed in vitro by the protection of unpaired thymines in the open complex against oxidation by KMnO4. Run-off transcription kinetics were also studied under identical conditions. Closure of the open promoter appears to be by far the rate-limiting step of transcription initiation and elongation for the linearized beta-lactamase gene, and is strongly dependent on template topology for the RNAI gene. It is suggested that the corresponding signals are deposited 30 bases at least downstream of transcription initiation and that promoter closure, and its clearance by elongating RNA polymerase, may occur almost simultaneously.

Base Sequence↗
PubMed1993

[Stability of messenger RNA of Escherichia coli ompA is affected by the use of synonymous codon].

The ompA gene of E. coli was silently mutated by the substitution of nine codons located towards the end of the gene, by synonymous codons expected to be translated more slowly. At 37 degrees C in vivo the life-time of the ompA messenger RNA (mRNA) is reduced from 4.5 min (w.t.) to 3.8 min (silent mutant). The amount of mRNA of the silent mutant is only 30% of that observed for the w.t. gene. These variations are thought to be due to the uncoupling of transcription from translation, and a lesser protection of the mRNA towards RNase digestion by ribosomes, resulting from a lesser density of the ribosome traffic on the mutated polysome.

Blotting, Northern↗
PubMed1993

The collision of cotranscribing E. coli RNA polymerases studied in vitro.

The collision of cotranscribing E. coli ternary complexes was brought about in vitro by forcing the first "leading" complex to stall before first template C, due to the absence of CTP, and then permitting initiation of a second "following" complex. Following collision, the transcript of the leading complex was increased in length by as much as 7 nt., despite the absence of CTP, but did not dissociate. Upon addition of CTP, the leading complex aborted at exactly the positions requiring incorporation of the next Cs', the following complex continued transcription. The observations point to the importance of linking appropriate promoter efficiency with transcriptional pausing times, and the role of transcriptional collisions in termination events.

Adenosine Triphosphate↗
PubMed1993

Transcription pausing signal detected by sense/antisense transcription.

We studied elongation pausing during transcription in vitro on both the sense and the antisense strands of a given gene fragment(5' end of the bla gene of Tn3). The average transcription rate on the sense strand was much lower than that of the antisense strand, and several pauses observed on the former have no detectable antisense partner. A pausing signal was identified, associated with an (AT)6 sequence, in the vicinity of a strong pause on the sense strand and the sole detected pause on the antisense strand. Mutation of this sequence strongly reduced, by a common factor, pausing times at both sites.

Base Sequence↗
PubMed1992

In vivo control of promoter and terminator efficiencies at a distance.

In pBR329, the genes providing resistance to ampicillin (beta-lactamase, bla) and chloramphenicol (chloramphenicol acetyl transferase, cat) are encoded on the same strand. The bla gene lies downstream of the cat gene, separated by an intergenic sequence of 414 bp. The transcription starts of the two genes are 1090 bp apart. We have probed, in vivo, the effect on transcription of the bla gene, of the introduction, in front of the cat gene, of a series of synthetic promoters covering a large (over 60-fold) range of efficiency. The rising efficiency of the cat promoter has several important consequences for transcription of the bla gene. First, a strong (up to sevenfold) stimulation of the bla promoter is observed, together with a shift of the main bla transcription start site, 10 bp upstream. Furthermore, the relative efficiencies of the bla transcription terminators are reduced. Finally, because of a lesser relative efficiency of the cat transcription terminators as well, we observe enhanced intrusion into the bla gene of transcripts initiated at the cat promoter, some of them extending to the bla transcription terminator and beyond. The operon-like expression of the cat-bla gene tandem is controlled by the efficiency of the cat terminator, which in turn depends on that of the cat promoter. This demonstrates a direct link between the efficiencies of promoter and terminator. Upon inhibition of bacterial gyrase activity, i.e. relaxation of negative supercoiling action, bla expression increases sharply in pBR329, but remains almost unchanged in a plasmid (pBRGC-1) in which cat is under the control of a 6.5-fold stronger promoter. Therefore, under normal gyrase activity, the stimulation of the bla promoter in pBRGC-1 (relative to pBR329) appears to be linked to topological relaxation of its template in situ, in keeping with earlier in vitro observations. We propose that the relaxed state of pBRGC-1 in situ could be due to the decrease in the plasmid linking number, introduced by the 10-12 RNA polymerases that simultaneously transcribe the cat gene in that plasmid, compared with only one or two in pBR329. We find that the negative superhelical densities of both plasmids are almost identical when extracted from the cell. Therefore gyrase would not correct for the relaxed state of plasmid pBRGC-1 observed in situ.

Ampicillin Resistance↗
PubMed1991

Kinetics of the specific binding of a second RNA polymerase to the standard bacterial-transposon-Tn3 bla promoter complex.

It was shown previously, that at moderate excess of RNA polymerase (RNAP) relative to DNA, the complex of the bla promoter from bacterial transposon Tn3 with RNAP is locked in an inactive, heparin-resistant, isomeric state which is not that of the 'open' complex. This 1:1 isomer can accommodate a second RNAP, which becomes tightly and specifically bound just upstream of the first RNAP [Duval-Valentin & Reiss (1990) Mol. Microbiol. 4, 1465-1475]. Both the resulting 2:1 complex and its antecedent 1:1 complex formed at excess of RNAP are immediately and permanently inhibited for transcription initiation. Using the gel-retardation technique, we investigate here the kinetics of formation and decay of the 2:1 complex under various experimental conditions. The data are consistent with pseudo-first-order kinetics at moderate excess of RNAP. The salt-dependence of rate and equilibrium constants has been analysed within the framework of the theoretical model described by Lohman, Dehaseth & Record [(1978) Biophys. Chem. 8, 281-294]. It was found that the salt-dependence is consistent with the existence of a transient intermediate during formation of the 2:1 complex, which forms rapidly on the time scale of its isomerization to the final 2:1 complex. The intermediate is characterized by the release of about seven cations from the 1:1 complex, one additional cation being released upon its final isomerization. Formation of the 2:1 complex at high excess of RNAP becomes inhibited, probably as a result of a 'bumping' effect of the complex by the enzyme, also observed with several other promoters. We conclude that formation of the 2:1 complex closely mimics that of the standard 1:1 complex, except that the final isomerization step to an 'open' complex is lacking. A mechanism of the formation of the 2:1 complex and of its role in transcription regulation of constitutive promoter by RNAP is proposed.

DNA Transposable Elements↗
PubMed1991

Effect of respiratory syncytial virus infection on mice with protein malnutrition.

Respiratory syncytial virus (RSV) pulmonary infection was produced in BALB/c mice fed protein-deficient diets in an effort to understand the severity of viral pneumonia in infants in developing countries. As in previously published experiments with Sendai virus, animals on the deficient diet became clinically malnourished, and certain aspects of their cell-mediated immunity were altered. The course of RSV infection in protein-deprived mice was essentially identical to that in normally nourished animals. The titer of virus recovered from lung homogenates over time, as well as the histologic picture of bronchiolitis, were identical under all experimental conditions. This model, unlike that of Sendai virus infection, fails to demonstrate an effect of protein malnutrition on RSV infection.

Animals↗
PubMed1990

Transcription in vivo directed by consensus sequences of E.coli promoters: their context heavily affects efficiencies and start sites.

We studied in vivo transcription and gene expression directed by a series of synthetic sequences, bearing the consensus hexamer (CH) pair of E.coli promoters in various contexts. The results demonstrate that, for the contexts tested, the CH pair supports transcription activity and gene expression, whether the spacer linking them is AT or GC rich, or is as short as 14 bp or as large as 26 bp (standard size 17 bp). However, we find that the context influences transcription efficiency by as much as an order of magnitude, and is able to scatter transcription start sites over a region of as much as 30 bp, including start sites within a CH or even between the two sequences of the CH pair. The results demonstrate that, although the CH pair can be sufficient for directing transcription by E.coli RNAP, important determinants for promoter activity are at least in part contained in the context of the consensus sequences; they advocate a synergic interplay of signals borne by the CH pair and its context, extending over all parts of the promoter sequence. A two-step model is proposed, in which properly located consensus sequences provide RNAP with facilities required for stereospecific docking along the promoter sequence; the result would be a sharp change in the local environment of the double helix inducing local isothermal unwinding. The size of the loop (related to the AT constraint in the promoter) and the extend of the environmental change required for unwinding would determine the rate of transcriptionally competent complex formation, positioning and grouping of start sites.

Base Sequence↗
PubMed1990

How Escherichia coli RNA polymerase can negatively regulate transcription from a constitutive promoter.

We previously described the structures and functions of specific complexes between the bla promoter from Tn3 (present in pBR322) and RNA polymerase (RNAP), showing that, at excess RNAP, complexes can form in which one or two RNAPs bind to the same promoter (1:1 and 2:1 complexes) (Duval-Valentin and Ehrlich, 1988). We report here that the 2:1 complex cannot be detected below 25 degrees C; above that temperature, a 1:1 complex forms at a rate one order of magnitude faster than that of the 2:1 complex, and above 30 degrees C, the amounts of both species become equal for RNAP/promoter ratio r30 less than or equal to r less than or equal to 70. The 2:1 complex decays back to a 1:1 complex losing the last RNAP at a rate about three times that of the 1:1 complex decay. Functional assays of the complexes formed at excess RNAP show that both 1:1 and 2:1 complexes are immediately and permanently inhibited, even when the promoters are pre-incubated with ribonucleotide selections potentially enabling entrance into abortive cycling or formation of a stressed complex. We conclude that the inhibition step probably takes place in the complex formation pathway between RPi and RPo, at a novel stable intermediate isomer, RPj, formed above 25 degrees C. A possible mechanism of formation of the 2:1 complex is outlined. In vivo studies, in which r was modified by varying the bacterial growth rate, show a reduction of bla expression as r values are upshifted, specific to the bla promoter from Tn3.

DNA-Directed RNA Polymerases↗
PubMed1989

In vivo gene expression directed by synthetic promoter constructions restricted to the -10 and -35 consensus hexamers of E. coli.

Two synthetic DNA sequences, carrying no other known E. coli promoter element than the consensus hexamers (CH) TTGACA (CH-35) and TATAAT CH(-10), spaced by 17 bp, were inserted in pBR329, in a position enabling transcription of the complete Cmr gene. The region upstream of the Cmr transcription start was carefully cleared of w.t. promoter elements (full deletion of the wild type (w.t.) Cmr promoter upstream +2 and large portion of an upstream coding sequence). Both synthetic promoters, which differ only by the sequences of the spacers (non consensus, constrained in AT or GC) support in vivo high level Cmr gene expression. The GC rich spacer is associated with transcription start at the usual +1 position, but with the AT rich spacer, transcription starts at several places, mainly in CH(-10). Rearranged promoter sequences derived from the synthetic ones upon transformation with partly ligated plasmids, yield new insights on the role of the standard CH pair, the size of the spacer and the sequence downstream of CH(-10).

Base Sequence↗
PubMed1987

Sequence deduced physical properties in the D-loop region common to five vertebrate mitochondrial DNAs.

Some sequence-induced physical properties of the region of the replication origin in human, mouse, rat, ox and xenopus mitochondrial DNA have been studied: characteristic profiles of stability can be observed, a consensus pattern of hydrogen bond donor/acceptor associated to a symmetrical distribution of base roll angles variation is found upstream of the 5' ends of the D-loop strand. In spite of diversity, evolution has conserved the collective physical properties in parts of the origin of replication region suggesting specific functions for these non-coding sequences.

Animals↗
PubMed1986

An algorithm for studying cooperative transitions in DNA.

Cooperative transitions in DNA (B to Z, B to A, helix to coil, etc.) are known to depend strongly on nucleotide sequence. In general the change in free energy involved in the transition can be expressed as: delta G(seq) = 2RT log (sigma) where sigma is a factor arising from the free energy associated with boundaries of different conformations along the molecule. This formula allows to infer a general algorithm with which DNA sequences can be partitioned into well defined domains in which, under suitable conditions, base pairs change state cooperatively. The different partitions of the sequence that can be generated by varying the values of the physical parameters involved in the above formula, are shown to be embedded into a binary tree hierarchy. Application to a reliable prediction of Z-DNA antibody binding sites will be illustrated for the 0X174 genome. Possible biological implications are briefly discussed.

Antibodies, Antinuclear↗
PubMed1985

Specific and non specific Escherichia coli ribonucleic acid polymerase DNA complexes are not hydrodynamically equivalent in analytical band sedimentation.

We have measured the sedimentation coefficients (s) of different DNA molecules of a few thousand base bairs in the presence of increasing amounts of E. coli RNA polymerase under conditions where tight binding complexes are formed. The measured s does not increase linearly with n(n=RNA Polymerase/DNA molar ratio); the s vs n plot can be decomposed into two parts; first the increase in s is small until n reaches a value n0 approximately equal to the number of strong promoters of the DNA molecule under consideration, then when n greater than n0 the slope of s(n) is much higher. The observations are in agreement with a model which postulates that strong specific polymerase binding leads to an increase in frictional coefficient of the RNA Polymerase-DNA complex, while non specific(or less specific)RNAP binding leads to a contraction of the RNA Polymerase-DNA complexes.

Binding Sites↗
PubMed1985

In vitro transcription initiation from three different Escherichia coli promoters. Effect of supercoiling.

Transcription initiation from beta-lactamase, tetracycline resistance and RNA 1 promoters, present in plasmid pAT153, were studied employing the abortive initiation technique. Assays appear to be promoter-specific with supercoiled and linear templates. Supercoiling enhances the isomerization rate constant of the open RNA-polymerase--promoter complex formation. Results agree with the in vivo behaviour of the corresponding promoters, and allow us to propose a hypothesis about the effect of supercoiling on transcription initiation.

DNA, Superhelical↗
PubMed1985

Common features of polyomavirus mutants selected on PCC4 embryonal carcinoma cells.

The genomic rearrangements of six polyomavirus mutants selected on PCC4 embryonal carcinoma cells have been compared and their common characteristics pointed out. All mutants show a duplication which includes at least the adenovirus type 5 (Ad5) E1A-like enhancer core sequence plus a deletion of variable size and location. The presence of the second enhancer core sequence, the SV40-like enhancer, is not required for expression of the PyEC PCC4 phenotype. Two of these mutants are also able to express polyomavirus T antigen on F9 and LT1 cells. Multiadaptation seems to require the duplication of the Ad5 E1A-like core sequence, the maintenance of the SV40-like core sequence and a local change in DNA stability.

Animals↗
PubMed1984

Local stability involved in characterizing and controlling promoters in eukaryotes.

Eukaryotic promoters with known in vivo activities have been analysed for characteristic stability patterns. Correlation of transcription yield in promoter mutants with size and stability of individual domains of the promoter stability profile supports the conclusion that eukaryotic promoters are built up by at least three elements: a region enabling the transcription ("enhancer"), with a characteristic stability pattern; an activator domain, with high GC content, whose activator potential is controlled by the domain stability and length; a trap domain, with high AT content, setting the cap site. The activated enzyme undergoes a steady deactivation process, losing half of its activity upon moving 55 bases between the activator and the trap site.

Base Composition↗
PubMed1984

Promoter recognition and transcription initiation in E. coli.

Analysis of stability maps of sequences harbouring E. coli RNA polymerase promoters shows a characteristic splitting in homostable domains, despite the heterogeneity of the sequences. Correlation of stability maps with results from static approaches giving the contact points of the enzyme on promoters and functional studies employing abortive initiation assay allow us to propose a general mechanism for recognition of promoters and transcription start.

Base Composition↗