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.

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.

A simple polypyrimidine repeat acts as an artificial Rho-dependent terminator in vivo and in vitro.

In this paper, we present evidence that an efficient Rho-dependent terminator can be created by introducing a simple (AG/TC) n DNA repeat into a transcription unit. The Rho termination activity in vivo and in vitro is dependent on the length and the orientation of the insert. The transcription of at least 30 bp of the (AG/TC) n repeat in the orientation encoding the (rUrC) n sequence on the transcript leads to Rho-dependent termination at a downstream non-terminator site. Our results indicate that the high efficiency of this artificial Rho-dependent terminator is due to optimal interactions between the (rUrC) n RNA sequence and Rho protein. Thus, our findings strongly suggest that an adequate loading site is the primary determinant for Rho termination activity and provide a more defined system for future investigations.

High resolution mapping of E.coli transcription elongation complex in situ reveals protein interactions with the non-transcribed strand.

We have used chemical probes and UV light to perform a high resolution mapping of an Escherichia coli transcription elongation complex that was arrested in vivo by a protein readblock at a position distal to the promoter. The in situ probing data provide a precise picture of a constrained ternary complex in which the front edge of the polymerase is located at <6 bp from the catalytic center. Furthermore, our analyses reveal protein contacts with the non-transcribed strand within the arrested ternary complex. Thus, these results contribute substantially to the emerging view of a flexible transcription elongation complex in which the non-transcribed strand is an important regulatory element.

Hyper-negative template DNA supercoiling during transcription of the tetracycline-resistance gene in topA mutants is largely constrained in vivo.

The excess linking deficit of plasmid DNA from topoisomerase I-defective bacteria (topA mutants) results mainly from transcription and is commonly ascribed to unbalanced relaxation of transcription-induced twin-supercoiled domains. This defect is aggravated in genes for membrane-binding proteins (such as the tet gene) where anchoring of the transcription complex to the bacterial membrane is thought to enhance twin-domain partitioning. Thus, it is often assumed that the 'hyper-negative' linking difference of plasmid DNA from topA mutants reflects unconstrained, hyper-negative DNA supercoiling inside the cell. We tested the validity of this assumption in the present study. A DNA sequence that undergoes a gradual B to Z transition under increasing negative superhelical tension was used as a sensor of unconstrained negative supercoiling. Z-DNA formation was probed at a site upstream from the inducible pTac promoter fused either to the tet gene or to the gene for cytosolic chloramphenicol acetyl transferase (cat). Although plasmid DNA linking deficit increased more extensively in topA mutants following tet activation than following cat activation, no significant differences were observed in the extents to which the B to Z DNA transition is stimulated in the two cases. We infer that the excess linking deficit of the tet-containing plasmid DNA reflects constrained negative DNA supercoiling inside the cell.

The size of the topological domain modulates the B-Z transition of a (TG)n containing repeat.

Under negative superhelical stress, long (TG)n containing repeats experience a stepwise multiple B-Z transitions. We have investigated the effect of the plasmid size on this transitional behavior. A 66-bp (TG)n containing repeat from the 5'-untranscribed region of mouse ribosomal DNA was inserted in a 3-kb, a 6.5-kb and a 12.5-kb plasmids and its supercoil-driven B-Z transition was followed by OsO4 probing of topoisomer-populations. Our results show a clear correlation between the size of the topological domain and the extent of the region that converts cooperatively into Z-DNA at the initial transition.

Radiolytic signature of Z-DNA.

Ionizing radiations induce various damages in DNA via the hydroxyl radical OH. generated by the radiolysis of water. We compare here the radiosensitivity of B- and Z-DNA, by using a Z-prone stretch included in a plasmid. In the supercoiled plasmid, the stretch is in the Z-form, whereas it is in the B-form when the plasmid is relaxed. Frank strand breaks (FSB) and alkali-revealed breaks (ARB) were located and quantified using sequencing gel electrophoresis. We show that B- and Z-DNA have the same mean sensitivity towards radiolytic attack, for both FSB and ARB. Nevertheless, the guanine sites are more sensitive, and the cytosine sites less sensitive in Z- than in B-DNA, leading to a characteristic signature of the Z-form. The comparison of experiments with the outcome of a Monte Carlo simulation of OH. radical attack suggests that transfer of initial damage from a guanine base to its attached sugar or the adjacent 3' cytosine is more important in Z-DNA than in B-DNA.

Gradual and oriented B-Z transition in 5'-untranscribed region of mouse ribosomal DNA.

The structural transition of an alternating purine-pyrimidine sequence (CG)5(TG)28) from the 5'-untranscribed region of the mouse ribosomal DNA was analyzed by two-dimensional gel electrophoresis and chemical probes. The repeat undergoes a supercoil-dependent gradual and oriented B-Z transition. At a threshold level of negative supercoiling, a limited region of the repeat encompassing the (CG)5 motif converts cooperatively into Z-DNA. As the superhelical stress increases, the Z-structure propagates along the remaining part of the repeat by successive transitions until the full-length sequence is converted. By in situ OsO4 probing experiments, we show also that this (TG)n-containing repeat adopts the Z-structure in Escherichia coli.

Z-DNA as a probe for localized supercoiling in vivo.

Biological processes such as transcription are expected to generate local variations in DNA supercoiling. The existence of localized supercoiling was recently demonstrated in Escherichia coli by using the supercoil-driven B-to-Z transition as a superhelicity probe. This new methodology is described and its extension to other biological systems discussed.

Direct evidence for the effect of transcription on local DNA supercoiling in vivo.

The B-to-Z structural transition of varying lengths (74 to 14 base-pairs) of (CG) tracts has been used as a superhelicity probe to examine the local topological changes induced by transcription at defined genetic loci in vivo. The local-topology reporter sequences indicate that under steady-state transcription the region upstream from the promoter experiences an increase in negative supercoiling whereas the region downstream from the terminator displays a decrease in negative superhelicity. This result provides direct in vivo evidence for the notion that the translocation of an RNA polymerase elongation complex along the double-helical DNA generates positive supercoils in front of it and negative supercoils behind it. Also, this twin-supercoiled domain model was tested inside a transcribed region where a high degree of negative supercoiling generated by the passage of each individual RNA polymerase was detected. Hence, these data indicate that the induced supercoils are confined to the vicinity of each RNA polymerase complex in a multipolymerase system.

Interstrand cross-links are preferentially formed at the d(GC) sites in the reaction between cis-diamminedichloroplatinum (II) and DNA.

A DNA restriction fragment with convergent SP6 and T7 promoters has undergone reaction with cis-diamminedichloroplatinum(II) (cis-DDP) and was then used as a template for RNA synthesis in vitro. The T7 and SP6 RNA polymerases generate fragments of defined sizes. Analysis of the RNA fragments shows that the polymerases are mainly blocked at the level of the d(GG) and d(AG) sites and to a lesser extent at the level of the d(GC) sites. The adducts at the d(GC) sites are more resistant to cyanide ion attack than those at the major sites and are identified as interstrand cross-links. The formation of an interstrand cross-link between the N-7 atoms of two guanine residues at the d(GC) sites was further confirmed by chemical modifications.

Flanking AT-rich tracts cause a structural distortion in Z-DNA in plasmids.

The effect of neighboring AT-rich sequences on the right-handed B to left-handed Z transition was investigated in plasmids. The supercoil stabilized Z-DNA structure in (CG) tracts 36 and 40 base pairs (bp) in length revealed an unexpected conformational aberration at defined C residues proximal to one end (colL) when the inserts were bilaterally flanked by an 80% AT-rich segment (90 bp on one side and 331 bp on the other). The presence of the perturbed Z-conformation required (CG) stretches longer than 32 bp and bilateral flanking by the AT-rich tracts, since plasmids with the (CG) tracts unilaterally flanked had an orthodox Z-structure. The thermodynamics of the negative super-coil-induced transitions were influenced only slightly by the neighboring AT-rich regions. Hence, the nature of Z-conformations in plasmids is markedly influenced by intrinsic structural features of the (Pur-Pyr) tract and by seemingly modest changes in the properties of neighboring sequences over a distance of several helical turns.

Stabilization of Z DNA in vivo by localized supercoiling.

Biological processes such as transcription may generate domains of supercoiling on a circular DNA. The existence of these domains in Escherichia coli was investigated by the ability of different lengths of (CG) tracts, cloned upstream or downstream from the tetracycline resistance gene (tet) of pBR322, to adopt the Z structure in vivo. Segments as short as 12 base pairs adopt the Z form when cloned upstream from the tet gene (Eco RI site), whereas no Z DNA was detected when this sequence was cloned downstream (Sty I site), even with a 74-base pair (CG) tract that requires less supercoiling than shorter tracts for the B-Z transition. Hence the localized supercoil density in pBR322 can be as high as -0.038 and as low as -0.021 at different loci. These data demonstrate the existence of the Z structure for commonly found natural sequences and support the notion of domains of negative supercoiling in vivo.