The presence of an RNA:DNA hybrid that is prone to slippage promotes termination by T7 RNA polymerase.

Intrinsic termination signals for multisubunit bacterial RNA polymerases (RNAPs) encode a GC-rich stem-loop structure followed by a polyuridine [poly(U)] tract, and it has been proposed that steric clash of the stem-loop with the exit pore of the RNAP imposes a shearing force on the RNA in the downstream RNA:DNA hybrid, resulting in misalignment of the active site. The structurally unrelated T7 RNAP terminates at a similar type of signal (TΦ), suggesting a common mechanism for termination. In the absence of a hairpin (passive conditions), T7 RNAP slips efficiently in both homopolymeric A and U tracts, and we have found that replacement of the U tract in TΦ with a slippage-prone A tract still allows efficient termination. Under passive conditions, incorporation of a single G residue following a poly(U) tract (which is the situation during termination at TΦ) results in a "locked" complex that is unable to extend the transcript. Our results support a model in which transmission of the shearing force generated by steric clash of the hairpin with the exit pore is promoted by the presence of a slippery tracts downstream, resulting in alterations in the active site and the formation of a locked complex that represents an early step in the termination pathway.

Yeast DEAD box protein Mss116p is a transcription elongation factor that modulates the activity of mitochondrial RNA polymerase.

DEAD box proteins have been widely implicated in regulation of gene expression. Here, we show that the yeast Saccharomyces cerevisiae DEAD box protein Mss116p, previously known as a mitochondrial splicing factor, also acts as a transcription factor that modulates the activity of the single-subunit mitochondrial RNA polymerase encoded by RPO41. Binding of Mss116p stabilizes paused mitochondrial RNA polymerase elongation complexes in vitro and favors the posttranslocated state of the enzyme, resulting in a lower concentration of nucleotide substrate required to escape the pause; this mechanism of action is similar to that of elongation factors that enhance the processivity of multisubunit RNA polymerases. In a yeast strain in which the RNA splicing-related functions of Mss116p are dispensable, overexpression of RPO41 or MSS116 increases cell survival from colonies that were exposed to low temperature, suggesting a role for Mss116p in enhancing the efficiency of mitochondrial transcription under stress conditions.

Histone variant H2A.Z inhibits transcription in reconstituted nucleosomes.

The existence of histone nonallelic variants has been known for more than 30 years, but only recently have we acquired significant insights into their functions. Nucleosomes containing histone variants are nonrandomly distributed in genomes and may impart different biological functions to the relevant chromatin regions. We have used the model T7 RNA polymerase to transcribe reconstituted nucleosomes containing either canonical human recombinant histones or two histone variants, H2A.Z or H3.3, whose presence has been associated with active transcription. Remarkably, in contrast to canonical and H3.3-containing nucleosomes, H2A.Z-containing nucleosomes were refractive to transcription, with residual levels of transcription determined by the sequence of the underlying DNA template. To our knowledge, this is the first example of a nucleosome that is intrinsically untranscribable.

Multiple functions of yeast mitochondrial transcription factor Mtf1p during initiation.

Transcription of the yeast mitochondrial genome is carried out by an RNA polymerase (Rpo41p) that is related to single subunit bacteriophage RNA polymerases but requires an additional factor (Mtf1p) for initiation. In this work we show that Mtf1p is involved in multiple roles during initiation including discrimination of upstream base pairs in the promoter, initial melting of three to four base pairs around the site of transcript initiation, and suppression of nonspecific initiation. It, thus, appears that Mtf1p is functionally analogous to initiation factors of multisubunit RNA polymerases, such as sigma. Photocross-linking experiments reveal close proximity between Mtf1p and the promoter DNA and show that the C-terminal domain makes contacts with the template strand in the vicinity of the start site. Interestingly, Mtf1p is related to a class of RNA methyltransferases, suggesting an early evolutionary link between RNA synthesis and processing.

In a head-on collision, two RNA polymerases approaching one another on the same DNA may pass by one another.

Using a template that contains promoters for T3 and T7 RNA polymerases (RNAPs) in opposing orientations, and His-tagged derivatives of these RNAPs that allow immobilization on solid matrices, we have determined that a T7 elongation complex (EC) may be advanced past a halted T3 EC, and that after the collision the halted T3 EC may resume transcription. Since RNAPs moving in opposite directions use two different strands of the DNA as their templates, it seems likely that they manage to pass by one other by temporarily releasing their nontemplate strand while maintaining association with their template strand.

Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification.

The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP-protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP-TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP-mtRNAP fusion, pulled down associated proteins, and identified them by LC-MS-MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity.

The transition to an elongation complex by T7 RNA polymerase is a multistep process.

During the transition from an initiation complex to an elongation complex (EC), T7 RNA polymerase undergoes major conformational changes that involve reorientation of a "core" subdomain as a rigid body and extensive refolding of other elements in the 266 residue N-terminal domain. The pathway and timing of these events is poorly understood. To examine this, we introduced proline residues into regions of the N-terminal domain that become alpha-helical during the reorganization and changed the charge of a key residue that interacts with the RNA:DNA hybrid 5 bp upstream of the active site in the EC but not in the initiation complex. These alterations resulted in a diminished ability to make products >5-7 nt and/or a slow transition through this point. The results indicate that the transition to an EC is a multistep process and that the movement of the core subdomain and reorganization of certain elements in the N-terminal domain commence prior to promoter release (at 8-9 nt).

Multisubunit RNA polymerases melt only a single DNA base pair downstream of the active site.

To extend the nascent transcript, RNA polymerases must melt the DNA duplex downstream from the active site to expose the next acceptor base for substrate binding and incorporation. A number of mechanisms have been proposed to account for the manner in which the correct substrate is selected, and these differ in their predictions as to how far the downstream DNA is melted. Using fluorescence quenching experiments, we provide evidence that cellular RNA polymerases from bacteria and yeast melt only one DNA base pair downstream from the active site. These data argue against a model in which multiple NTPs are lined up downstream of the active site.

Template misalignment in multisubunit RNA polymerases and transcription fidelity.

Recent work showed that the single-subunit T7 RNA polymerase (RNAP) can generate misincorporation errors by a mechanism that involves misalignment of the DNA template strand. Here, we show that the same mechanism can produce errors during transcription by the multisubunit yeast RNAP II and bacterial RNAPs. Fluorescence spectroscopy reveals a reorganization of the template strand during this process, and molecular modeling suggests an open space above the polymerase active site that could accommodate a misaligned base. Substrate competition assays indicate that template misalignment, not misincorporation, is the preferred mechanism for substitution errors by cellular RNAPs. Misalignment could account for data previously taken as evidence for additional NTP binding sites downstream of the active site. Analysis of the effects of different template topologies on misincorporation indicates that the duplex DNA immediately downstream of the active site plays an important role in transcription fidelity.

A mechanism of nucleotide misincorporation during transcription due to template-strand misalignment.

Transcription errors by T7 RNA polymerase (RNAP) may occur as the result of a mechanism in which the template base two positions downstream of the 3' end of the RNA (the TSn+1 base) is utilized during two consecutive nucleotide-addition cycles. In the first cycle, misalignment of the template strand leads to incorporation of a nucleotide that is complementary to the TSn+1 base. In the second cycle, the template is realigned and the mismatched primer is efficiently extended, resulting in a substitution error. Proper organization of the transcription bubble is required for maintaining the correct register of the DNA template, as the presence of a complementary nontemplate strand opposite the TSn+1 base suppresses template misalignment. Our findings for T7 RNAP are in contrast to related DNA polymerases of the Pol I type, which fail to extend mismatches efficiently and generate predominantly deletion errors as a result of template-strand misalignment.

Single-molecule approaches reveal the idiosyncrasies of RNA polymerases.

Recently developed single-molecule techniques have provided new insights into the function of one of the most complex and highly regulated processes in the cell--the transcription of the DNA template into RNA. This review discusses methods and results from this emerging field, and it puts them in perspective of existing biochemical and structural data.

Probing conformational changes in T7 RNA polymerase during initiation and termination by using engineered disulfide linkages.

During the transition from an initiation complex to an elongation complex (EC), the single-subunit bacteriophage T7 RNA polymerase (RNAP) undergoes dramatic conformational changes. To explore the significance of these changes, we constructed mutant RNAPs that are able to form disulfide bonds that limit the mobility of elements that are involved in the transition (or its reversal) and examined the effects of the crosslinks on initiation and termination. A crosslink that is specific to the initiation complex conformation blocks transcription at 5-6 nt, presumably by preventing isomerization to an EC. A crosslink that is specific to the EC conformation has relatively little effect on elongation or on termination at a class I terminator (T), which involves the formation of a stable stem-loop structure in the RNA. Crosslinked ECs also pause and resume transcription normally at a class II pause site (concatamer junction) but are deficient in termination at a class II terminator (PTH, which is found in human preparathyroid hormone gene), both of which involve a specific recognition sequence. The crosslinked amino acids in the EC lie close to the upstream end of the RNA-DNA hybrid and may prevent a movement of the polymerase that would assist in displacing or releasing RNA from a relatively unstable DNA-RNA hybrid in the paused PTH complex.

A tightly regulated molecular motor based upon T7 RNA polymerase.

Controlled movement of materials or molecules within the nanometer range is essential in many applications of nanotechnology. Here we report the capture, movement, and release of cargo molecules along DNA by a modified form of T7 RNA polymerase (RNAP) in a manner that is controlled by the sequence of the DNA. Using single-molecule methods, we visualize the assembly and manipulation of nanodevices and the ability to harness rotary and linear forces of the RNAP motor.

RNA displacement and resolution of the transcription bubble during transcription by T7 RNA polymerase.

Unlike DNA polymerases, RNA polymerases (RNAPs) must displace the nascent product from the template and restore the DNA to duplex form after passage of the transcription complex. To accomplish this, RNAPs establish a locally denatured "bubble" that encloses a short RNA:DNA hybrid. As the polymerase advances along the template, the RNA is displaced at the trailing edge of the bubble and the two DNA strands are reannealed. Structural analyses have revealed a number of elements that are likely to be involved in this process in T7 RNAP. In this work, we used genetic and biochemical methods to explore the roles of these elements during the transition from an initiation complex to an elongation complex. The results indicate that the transition is a multistep process and reveal a critical role for the nontemplate strand of the DNA.

Structural basis for substrate selection by t7 RNA polymerase.

The mechanism by which nucleotide polymerases select the correct substrate is of fundamental importance to the fidelity of DNA replication and transcription. During the nucleotide addition cycle, pol I DNA polymerases undergo the transition from a catalytically inactive "open" to an active "closed" conformation. All known determinants of substrate selection are associated with the "closed" state. To elucidate if this mechanism is conserved in homologous single subunit RNA polymerases (RNAPs), we have determined the structure of T7 RNAP elongation complex with the incoming substrate analog. Surprisingly, the substrate specifically binds to RNAP in the "open" conformation, where it is base paired with the acceptor template base, while Tyr639 provides discrimination of ribose versus deoxyribose substrates. The structure therefore suggests a novel mechanism, in which the substrate selection occurs prior to the isomerization to the catalytically active conformation. Modeling of multisubunit RNAPs suggests that this mechanism might be universal for all RNAPs.

Exposure of T7 RNA polymerase to the isolated binding region of the promoter allows transcription from a single-stranded template.

While the binding region of the T7 promoter must be double-stranded (ds) to function, the non-template strand in the initiation region is dispensable, and a promoter that lacks this element allows efficient initiation. To determine whether the binding region serves merely to recruit the RNA polymerase (RNAP) to the vicinity of a melted initiation region or provides other functions, we utilized a GAL4-T7 RNAP fusion protein to provide an independent binding capacity to the RNAP. When the GAL4-T7 RNAP was recruited to a single-stranded (ss) promoter via a nearby Gal4 recognition sequence, no transcription was observed. However, transcription from the ss promoter could be activated by the addition, in trans, of a ds hairpin loop that contains only the binding region of the promoter. The same results were obtained in the absence of the GAL4 recognition sequence in the template and were also observed with wild type enzyme. Gel-shift experiments indicate that exposure of the RNAP to the isolated binding region facilitates recruitment of the ss template, but that the binding region is displaced from the complex prior to initiation. We conclude that exposure of the RNAP to the isolated binding region reorganizes the enzyme, allowing it to bind to the ss template. These findings have potential implications with regard to mechanisms of promoter binding and melting.

Crystallization and preliminary crystallographic analysis of T7 RNA polymerase elongation complex.

Stable transcription-elongation complexes consisting of T7 RNA polymerase (molecular mass 99 kDa) in association with a nucleic acid scaffold consisting of an 8 bp RNA-DNA hybrid and 10 bp of downstream DNA were assembled and crystallized by the sitting-drop vapour-diffusion technique under near-physiological conditions. The crystals diffract beyond 2.6 A resolution and belong to space group P1, with unit-cell parameters a = 79.91, b = 84.97, c = 202 A, alpha = 90.36, beta = 92.97, gamma = 109.94 degrees. An unambiguous molecular-replacement solution was found using the C-terminal portion of the T7 RNA polymerase structure from the early initiation complex as a search model. Model building and structure refinement are now in progress.

Structure of a T7 RNA polymerase elongation complex at 2.9 A resolution.

The single-subunit bacteriophage T7 RNA polymerase carries out the transcription cycle in an identical manner to that of bacterial and eukaryotic multisubunit enzymes. Here we report the crystal structure of a T7 RNA polymerase elongation complex, which shows that incorporation of an 8-base-pair RNA-DNA hybrid into the active site of the enzyme induces a marked rearrangement of the amino-terminal domain. This rearrangement involves alternative folding of about 130 residues and a marked reorientation (about 130 degrees rotation) of a stable core subdomain, resulting in a structure that provides elements required for stable transcription elongation. A wide opening on the enzyme surface that is probably an RNA exit pathway is formed, and the RNA-DNA hybrid is completely buried in a newly formed, deep protein cavity. Binding of 10 base pairs of downstream DNA is stabilized mostly by long-distance electrostatic interactions. The structure implies plausible mechanisms for the various phases of the transcription cycle, and reveals important structural similarities with the multisubunit RNA polymerases.

Characterization of T7 RNA polymerase transcription complexes assembled on nucleic acid scaffolds.

We have used synthetic oligomers of DNA and RNA to assemble nucleic acid scaffolds that, when mixed with T7 RNA polymerase, allow the formation of functional transcription complexes. Manipulation of the scaffold structure allows the contribution of each element in the scaffold to transcription activity to be independently determined. The minimal scaffold that allows efficient extension after challenge with 200 mm NaCl consists of an 8-nt RNA primer hybridized to a DNA template (T strand) that extends 5-10 nt downstream. Constructs in which the RNA-DNA hybrid is less than or greater than 8 bp are less salt-resistant, and the hybrid cannot be extended beyond 12-13 bp. Although the presence of a complementary nontemplate strand downstream of the primer does not affect salt resistance, the presence of DNA upstream decreases resistance. The addition of a 4-nt unpaired "tail" to the 5' end of the primer increases salt resistance, as does the presence of an unpaired nontemplate strand in the region that contains the 8-bp hybrid (thereby generating an artificial transcription "bubble"). Scaffold complexes having these features remain active for over 1 week in the absence of salt and exhibit many of the properties of halted elongation complexes, including resistance to salt challenge, a similar trypsin cleavage pattern, and a similar pattern of RNA-RNA polymerase cross-linking.