Overextended RNA:DNA hybrid as a negative regulator of RNA polymerase II processivity.

An eight nucleotide RNA:DNA hybrid at the 3' end of the transcript is required for the stability of the elongation complex (EC) of RNA polymerase II. A non-template DNA strand is not needed for the stability of the EC, which contains this minimal hybrid. Here, we apply a recently developed method for promoter-independent assembly of functional EC of RNA polymerase II from synthetic RNA and DNA oligonucleotides to study the minimal composition of the nucleic acid array required for stability of the complex with RNA longer than eight nucleotides. We found that upon RNA extension beyond 14-16 nt in the course of transcription, non-template DNA becomes essential for maintaining a stable EC. Our data suggest that the overextended RNA:DNA hybrid formed in the absence the non-template DNA acts as a negative regulator of EC stability. The dissociation of the EC correlates with the backsliding of the polymerase along the overextended hybrid. The dual role of the hybrid provides a mechanism for the control of a correct nucleic acid architecture in the EC and of RNA polymerase II processivity.

The 8-nucleotide-long RNA:DNA hybrid is a primary stability determinant of the RNA polymerase II elongation complex.

The sliding clamp model of transcription processivity, based on extensive studies of Escherichia coli RNA polymerase, suggests that formation of a stable elongation complex requires two distinct nucleic acid components: an 8-9-nt transcript-template hybrid, and a DNA duplex immediately downstream from the hybrid. Here, we address the minimal composition of the processive elongation complex in the eukaryotes by developing a method for promoter-independent assembly of functional elongation complex of S. cerevisiae RNA polymerase II from synthetic DNA and RNA oligonucleotides. We show that only one of the nucleic acid components, the 8-nt RNA:DNA hybrid, is necessary for the formation of a stable elongation complex with RNA polymerase II. The double-strand DNA upstream and downstream of the hybrid does not affect stability of the elongation complex. This finding reveals a significant difference in processivity determinants of RNA polymerase II and E. coli RNA polymerase. In addition, using the imperfect RNA:DNA hybrid disturbed by the mismatches in the RNA, we show that nontemplate DNA strand may reduce the elongation complex stability via the reduction of the RNA:DNA hybrid length. The structure of a "minimal stable" elongation complex suggests a key role of the RNA:DNA hybrid in RNA polymerase II processivity.

Oriented, active Escherichia coli RNA polymerase: an atomic force microscope study.

Combining a system for binding proteins to surfaces (Sigal, G. B., C. Bamdad, A. Barberis, J. Strominger, and G. M. Whitesides. 1996. Anal. Chem. 68:490-497) with a method for making ultraflat gold surfaces (Hegner, M., P. Wagner, and G. Semenza. 1993. Surface Sci. 291:39-46 1993) has enabled single, oriented, active Escherichia coli RNA polymerase (RNAP) molecules to be imaged under aqueous buffer using tapping-mode atomic force microscopy (AFM). Recombinant RNAP molecules containing histidine tags (hisRNAP) on the C-terminus were specifically immobilized on ultraflat gold via a mixed monolayer of two different omega-functionalized alkanethiols. One alkanethiol was terminated in an ethylene-glycol (EG) group, which resists protein adsorption, and the other was terminated in an N-nitrilotriacetic acid (NTA) group, which binds the histidine tag through two coordination sites with a nickel ion. AFM images showed that these two alkanethiols phase-segregate. Specific binding of the hisRNAP molecules was followed in situ by injecting proteins directly into the AFM fluid cell. The activity of the hisRNAP bound to the NTA groups was confirmed with a 42-base circular single-stranded DNA template (rolling circle), which the RNAP uses to produce huge RNA transcripts. These transcripts were imaged in air after the samples were rinsed and dried, since RNA also has low affinity for the EG-thiol and cannot be imaged under the buffers we used.

Functional topography of nascent RNA in elongation intermediates of RNA polymerase.

To determine the dynamics of transcript extrusion from Escherichia coli RNA polymerase (RNAP), we used degradation of the RNA by RNases T1 and A in a series of consecutive elongation complexes (ECs). In intact ECs, even extremely high doses of the RNases were unable to cut the RNA closer than 14-16 nt from the 3' end. Our results prove that all of the cuts detected within the 14-nt zone are derived from the EC that is denatured during inactivation of the RNases. The protected zone monotonously translocates along the RNA after addition of new nucleotides to the transcript. The upstream region of the RNA heading toward the 5' end is cleaved and dissociated from the EC, with no effect on the stability and activity of the EC. Most of the current data suggest that an 8- to 10-nt RNA.DNA hybrid is formed in the EC. Here, we show that an 8- to 10-nt RNA obtained by truncating the RNase-generated products further with either GreB or pyrophosphate is sufficient for the high stability and activity of the EC. This result suggests that the transcript-RNAP interaction that is required for holding the EC together can be limited to the RNA region involved in the 8- to 10-nt RNA.DNA hybrid.

Crucial role of the RNA:DNA hybrid in the processivity of transcription.

We present an approach for studying the role of complementary nucleic acid interactions in transcription elongation by E. coli RNA polymerase (RNAP). The method involves in vitro reconstitution of a catalytically active elongation complex (EC) by the addition of RNAP to a single-strand DNA oligonucleotide containing the preannealed RNA primer, followed by incorporation of the complementary nontemplate DNA oligonucleotide. In all parameters tested, the reconstituted complex is indistinguishable from normal EC obtained by promoter-specific initiation. Using RNA primers of different lengths, which were fully or partially complementary to the DNA, we determined the minimal transcript length and the degree of its template pairing that is required to stabilize protein/ nucleic acid interactions in EC to the high level characteristic of normal transcription. Our data show that a hybrid at least 9 nt long, formed between the template DNA and 3'-proximal RNA transcript, is necessary for the high processivity of EC during RNA chain elongation.

RNA polymerase switches between inactivated and activated states By translocating back and forth along the DNA and the RNA.

Important regulatory events in both prokaryotic and eukaryotic transcription are currently explained in terms of an inchworming model of elongation. In this model, RNA extension is carried out by a mobile catalytic center that, at certain DNA sites, advances within stationary RNA polymerase. This idea emerged from the observation that footprints of individual elongation complexes, halted in vitro at consecutive DNA positions, can remain fixed on the template for several contiguous nucleotide additions. Here, we examine in detail the structural transitions that occur immediately after the enzyme stops at sites where discontinuous advancement of RNA polymerase is observed. We demonstrate that halting at such special sites does not "freeze" RNA polymerase at one location but induces it to leave its initial position and to slide backward along the DNA and the RNA without degrading the transcript. The resulting loss of contact between the RNA 3'-hydroxyl and the enzyme's catalytic center leads to temporary loss of the catalytic activity. This process is equilibrated with enzyme return to the original location, so that RNA polymerase is envisaged as an oscillating object switching between catalytically active and inactive states. The retreated isoform constitutes a principal intermediate in factor-induced endonucleolytic RNA cleavage. These oscillations of RNA polymerase can explain its apparent discontinuous advancement, which had been interpreted as indicating flexibility within the enzyme.

Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3' end of the RNA intact and extruded.

RNA polymerase (RNAP) may become arrested during transcript elongation when ternary complexes remain intact but further RNA synthesis is blocked. Using a combination of DNA and RNA footprinting techniques, we demonstrate that the loss of catalytic activity upon arrest of Escherichia coli RNAP is accompanied by an isomerization of the ternary complex in which the enzyme disengages from the 3' end of the transcript and moves backward along the DNA with concomitant reverse threading of the intact RNA through the enzyme. The reversal of RNAP brings the active center to the internal RNA position and thereby it represents a step in factor-facilitated transcript cleavage. Secondary structure elements or the 5' end of the transcript can prevent the isomerization by blocking the RNA threading. The described novel property of RNAP has far-reaching implications for the understanding of the elongation mechanism and gene regulation.

Molecular cloning and characterization of Borrelia burgdorferi rpoB.

Borrelia burgdorferi rpoB, the gene encoding the beta-subunit of RNA polymerase, has been cloned and sequenced. The full-length gene encodes a protein of 1154 amino acids with a calculated molecular mass of 129.8 kDa. The amino-acid sequence is 49% identical to the corresponding protein from Escherichia coli. B. burgdorferi rpoB is a component of a gene cluster, which includes rplJ, rplL and rpoC. A temperature-sensitive E. coli rpoB mutant could be complemented by introduction of the B. burgdorferi gene, indicating that the B. burgdorferi rpoB is expressed in E. coli and the beta-subunit can be assembled into functional holoenzyme. The wild-type amino-acid sequence of the B. burgdorferi beta-subunit is consistent with those of spontaneously arising rifampicin-resistant mutants of E. coli and Mycobacterium tuberculosis at certain critical residues. This suggests that the natural resistance of B. burgdorferi to rifampicin may be due to the primary amino-acid sequence of its beta-subunit.

Escherichia coli RNA polymerase activity observed using atomic force microscopy.

Fluid tapping-mode atomic force microscopy (AFM) was used to observe Escherichia coli RNA polymerase (RNAP) transcribing two different linear double-stranded (ds) DNA templates. The transcription process was detected by observing the translocation of the DNA template by RNAP on addition of ribonucleoside 5'-triphosphates (NTPs) in sequential AFM images. Stalled ternary complexes of RNAP, dsDNA and nascent RNA were adsorbed onto a mica surface and imaged under continuously flowing buffer. On introduction of all four NTPs, we observed some DNA molecules being pulled through the RNAP, some dissociating from the RNAP and others which did not move relative to the RNAP. The transcription rates were observed to be approximately 0.5-2 bases/s at our NTP concentrations, approximately 5 microM. The RNA transcripts were not unambiguously imaged in fluid. However, in experiments using a small single-stranded (ss) circular DNA template, known as a rolling circle, transcripts up to 1 or 2 microns long could be observed with tapping mode AFM once the samples were dried and imaged in air. This confirmed our observations of the transcriptional activity of RNAP adsorbed onto mica. This work illustrates that the development of tapping-mode in fluid has made it possible to use AFM to follow biological processes at the molecular level and get new insights about the variability of activity of individual molecules bound to a surface.

Mapping of catalytic residues in the RNA polymerase active center.

When the Mg2+ ion in the catalytic center of Escherichia coli RNA polymerase (RNAP) is replaced with Fe2+, hydroxyl radicals are generated. In the promoter complex, such radicals cleave template DNA near the transcription start site, whereas the beta' subunit is cleaved at a conserved motif NADFDGD (Asn-Ala-Asp-Phe-Asp-Gly-Asp). Substitution of the three aspartate residues with alanine creates a dominant lethal mutation. The mutant RNAP is catalytically inactive but can bind promoters and form an open complex. The mutant fails to support Fe2+-induced cleavage of DNA or protein. Thus, the NAD-FDGD motif is involved in chelation of the active center Mg2+.

Coupling between transcription termination and RNA polymerase inchworming.

Advancement of RNA polymerase of E. coli occurs in alternating laps of monotonic and inchworm-like movement. Cycles of inchworming are encoded in DNA and involve straining and relaxation of the ternary complex accompanied by characteristic leaping of DNA and RNA footprints. We demonstrate that the oligo(T) tract that constitutes a normal part of transcription terminators acts as an inchworming signal so that the leap coincides with the termination event. Prevention of leaping with a roadblock of cleavage-defective EcoRI protein results in suppression of RNA chain release at a termination site. The results indicate that straining and relaxation of RNA polymerase are steps in the termination mechanism.

Assembly of functional Escherichia coli RNA polymerase containing beta subunit fragments.

The Escherichia coli rpoB gene, which codes for the 1342-residue beta subunit of RNA polymerase (RNAP), contains two dispensable regions centered around codons 300 and 1000. To test whether these regions demarcate domains of the RNAP beta subunit, fragments encoded by segments of rpoB flanking the dispensable regions were individually overexpressed and purified. We show that these beta-subunit polypeptide fragments, when added with purified recombinant beta', sigma, and alpha subunits of RNAP, reconstitute a functional enzyme in vitro. These results demonstrate that the beta subunit is composed of at least three distinct domains and open another avenue for in vitro studies of RNAP assembly and structure.

Topology of the RNA polymerase active center probed by chimeric rifampicin-nucleotide compounds.

Spatial organization of the binding sites for the priming substrate, the template DNA, and the transcription inhibitor rifampicin (Rif) in Escherichia coli RNA polymerase (EC 2.7.7.6) was probed with chimeric compounds in which Rif is covalently attached to a ribonucleotide. The compounds bind to RNA polymerase in bifunctional manner and serve as substrates for RNA chain extension, yielding chains up to 8 nucleotides in length, with Rif linked to their 5' termini. These products act as potent inhibitors of normal transcription. Using the linker between the two ligands as ruler, we determined the distance between the sites for Rif and the priming nucleotide to be approximately 15 A. A reactive side group placed in the linker next to Rif crosslinks to the template strand of DNA at the -2 or -3 position of the promoter. Thus, bound Rif is juxtaposed to DNA immediately upstream of the start site, suggesting that Rif plugs the channel leading RNA out of the active center.

Motion and enzymatic degradation of DNA in the atomic force microscope.

The dynamics and enzymatic degradation of single DNA molecules can now be observed with the atomic force microscope. A combination of two advances has made this possible. Tapping in fluid has reduced lateral forces, which permits the imaging of loosely adsorbed molecules; and the presence of nickel ions appears to form a relatively stable bridge between the negatively charged mica and the negatively charged DNA phosphate backbone. Continuous imaging shows DNA motion and the process of DNA degradation by the nuclease DNase I. It is possible to see DNase degradation of both loosely adsorbed and tightly adsorbed DNA molecules. This method gives images in aqueous buffer of bare, uncoated DNA molecules with lengths of only a few hundred base pairs, or approximately 100 nm in length.

Discontinuous mechanism of transcription elongation.

During transcription elongation, three flexibly connected parts of RNA polymerase of Escherichia coli advance along the template so that the front-end domain is followed by the catalytic site which in turn is followed by the RNA product binding site. The advancing enzyme was found to maintain the same conformation throughout extended segments of the transcribed region. However, when the polymerase traveled across certain DNA sites that seemed to briefly anchor the front-end domain, cyclic shifting of the three parts, accompanied by buildup and relief of internal strain, was observed. Thus, elongation proceeded in alternating laps of monotonous and inchworm-like movement with the flexible RNA polymerase configuration being subject to direct sequence control.

A non-essential domain of Escherichia coli RNA polymerase required for the action of the termination factor Alc.

An evolutionarily nonconserved region of approximately 250 amino acids can be deleted from the amino-terminal part of the beta subunit of Escherichia coli RNA polymerase without effect on the enzyme's basic function. The non-essential segment is located between two highly conserved motifs and is flanked by sequences participating in the rifampicin-binding site. The results define the second non-essential domain in the beta subunit, in addition to the more distal dispensable segment identified previously. The Alc protein of bacteriophage T4 participates in the host transcription shutoff after infection by causing premature termination of transcription on E. coli DNA. Point mutations which prevent Alc action in vivo change amino acids in the non-essential NH2-terminal domain of the beta subunit. These point mutations as well as deletions which remove the non-essential region also prevent Alc action. Thus, in the RNA polymerase molecule, the proximal non-essential domain of beta may function as an acceptor of Alc or other regulatory factors.

Bacteriophage T4 Alc protein: a transcription termination factor sensing local modification of DNA.

Bacteriophage T4 Alc protein participates in shutting off host transcription after infection of E. coli. It is demonstrated that Alc acts as a site-specific termination factor. The Alc sites occur frequently in E. coli DNA, resulting in early cessation of elongation in several tested transcription units. Alc-dependent termination requires unimpeded propagation of the elongating complex as it approaches the Alc site. Temporary halting of RNA polymerase within 10-15 bp before the Alc site prevents termination. Bacteriophage T4 transcription is protected from the action of Alc by overall substitution of cytosine with 5-hydroxymethyl cytosine in T4 DNA. In vitro methylation of CpG sequences in the vicinity of an Alc site abolishes the effect of Alc. Thus, Alc-dependent termination involves local sensing of the state of cytosine modification and a short-term "memory" of recent pausing.