Francine Toulme

Transcription through the roadblocks: the role of RNA polymerase cooperation

During transcription, cellular RNA polymerases (RNAP) have to deal with numerous potential roadblocks imposed by various DNA binding proteins. Many such proteins partially or completely interrupt a single round of RNA chain elongation in vitro. Here we demonstrate that Escherichia coli RNAP can effectively read through the site‐specific DNA‐binding proteins in vitro and in vivo if more than one RNAP molecule is allowed to initiate from the same promoter. The anti‐roadblock activity of the trailing RNAP does not require transcript cleavage activity but relies on forward translocation of roadblocked complexes. These results support a cooperation model of transcription whereby RNAP molecules behave as ‘partners’ helping one another to traverse intrinsic and extrinsic obstacles.

GreA and GreB proteins revive backtracked RNA polymerase in vivo by promoting transcript trimming.

The GreA and GreB proteins of Escherichia coli show a multitude of effects on transcription elongation in vitro, yet their physiological functions are poorly understood. Here, we investigated whether and how these factors influence lateral oscillations of RNA polymerase (RNAP) in vivo, observed at a protein readblock. When RNAP is stalled within an (ATC/TAG)n sequence, it appears to oscillate between an upstream and a downstream position on the template, 3 bp apart, with concomitant trimming of the transcript 3′ terminus and its re‐synthesis. Using a set of mutant E.coli strains, we show that the presence of GreA or GreB in the cell is essential to induce this trimming. We show further that in contrast to a ternary complex that is stabilized at the downstream position, the oscillating complex relies heavily on the GreA/GreB‐induced ‘cleavage‐and‐restart’ process to become catalytically competent. Clearly, by promoting transcript shortening and re‐alignment of the catalytic register, the Gre factors function in vivo to rescue RNAP from being arrested at template positions where the lateral stability of the ternary complex is impaired.

Photosensitized splitting of thymine dimers in DNA by gene 32 protein from phage T 4.

Abstract The binding of denatured DNA to the protein coded by gene 32 of phage T 4 is accompanied by a quenching of the fluorescence of the protein tryptophyl residues. Gene 32 protein also binds to UV-irradiated DNA and photosensitizes the splitting of thymine dimers. Thymine bases are regenerated by this photosensitized reaction both in double stranded and in heat denatured DNA. No photosensitized splitting of thymine dimers is observed when the complex formed by gene 32 protein with UV-irradiated DNA is dissociated at high ionic strength. These results are discussed with respect to the possible stacking interaction of tryptophyl residues of gene 32 protein with bases in single stranded DNA.

In vivo evidence for back and forth oscillations of the transcription elongation complex

We have used a combination of DNA and RNA footprinting experiments to analyze the structural rearrangements experienced by a transcription elongation complex that was halted in vivo by a protein readblock. We show that the complex readblocked within an (ATC/TAG)n sequence is in a dynamic equilibrium between upstream‐ and downstream‐ translocated conformers. By increasing the strength of the putative RNA‐DNA hybrid, the ternary complex is readily trapped in the downstream‐translocated conformation, where the melted DNA region is limited to 8 bp. The shift of the equilibrium towards the downstream location is also achieved by introducing within the 5′ end of the message an RNA sequence that can pair with a segment of the transcript in the vicinity of the halted ternary complex. Our results demonstrate that within certain template DNA sequences, the back and forth oscillations of the ternary complex actually occur in a multipolymerase system and inside the cell. Furthermore, the cis‐acting effect of the upstream RNA sequence underscores an important phenomenon in gene regulation where a transcript may regulate its own elongation.

Structure and Dynamics of Peptide-Nucleic Acid Complexes.

Understanding the molecular mechanisms which govern the selective association of proteins with nucleic acids requires a detailed knowledge of the interactions involving the functional groups of both molecules (1). These interactions can be better characterized at the level of simple systems such as oligopeptide-oligonucleotide complexes even though extrapolation of these results to real biological systems may raise difficulties. However it is hoped that the physico-chemical characteristics of defined interactions between nucleic acid bases and amino acid side chains will help identify them in protein-nucleic acid complexes. A number of studies have been devoted to oligopeptide-nucleic acid interactions during the past ten years (for a recent review see reference 2). In this paper we further characterize electrostatic and stacking interactions involved in the binding of oligopeptides containing basic and aromatic residues to nucleic acids.

PHOTOSENSITIZED SPLITTING OF THYMINE DIMERS IN DNA BY PEPTIDES AND PROTEIN CONTAINING TRYPTOPHANYL RESIDUES

ABSTRACT Thymine dimers formed in DNA by UV irradiation can be split by irradiation at 300 nm in the presence of photosensitizers containing an indole ring : serotonin and 5-methoxytryptamine, an oligopeptide containing a tryptophyl residue (Lys-Trp-Lys), and a protein which exhibits a specificity for single stranded nucleic acids (protein coded for by gene 32 of phage T4). The mechanism of this photosensitized reaction has been investigated using different methods : fluorescence spectroscopy at room and at low temperature, flash photolysis, effect of electron scavengers, ionic strength dependence. In order to act as a photosensitizer, the indole-containing compound must be bound to UV irradiated DNA and stacked with nucleic acid bases and thymine dimers. Dissociation of the complex inhibits the reaction. The splitting arises as a consequence of an electron transfer from the excited indole ring to the dimer. In all cases, the splitting of thymine dimers regenerates intact thymine bases.

PHOTOCHEMICAL REACTIONS OF THYMINE WITH CARBOXYLIC ACIDS. A MODEL FOR NUCLEIC ACID-PROTEIN PHOTOCROSSLINKING

When nucleic acid bases are UV‐irradiated in the presence of carboxylic acids or carboxylate anions new photoproducts are formed as compared to the bases irradiated in the absence of carboxylic acids. The behavior of thymine and thymidine has been examined in detail. At least four photoproducts are formed in the presence of propionic acid and three in the presence of butyric acid. None of them appears to be a cyclobutyl dimer. From the concentration dependence of the rate of photoproduct formation it is concluded that the reactive excited species is the first excited singlet state of thymine. When 14C‐labelled thymine is irradiated in the presence of polyglutamic acid an important part of the radioactive material is covalently linked to the polymer. Photochemical reaction of thymine with glutamic (or aspartic) acid could thus induce crosslinks between proteins and nucleic acids. It is also shown that these photoproducts are stable under usual conditions of acidic hydrolysis of UV‐irradiated DNA.