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The in vitro studies of three T7 RNA polymerase point mutants suggest that substitutions of Ala and Thr for Pro-563 and of Ser for Tyr-571 have little effect on the enzyme catalytic competence, but result in its inability to utilize the promoter. Both P563A and P563T mutants retain the promoter-binding ability, whereas the promoter affinity of the Y571S mutant drops drastically.
The protein hRPABC14.4 is an essential subunit of human RNA polymerases I, II, and III and is required for the transcription of all human nuclear genes. The structure of hRPABC14.4 was determined by nuclear magnetic resonance spectroscopy. The protein fold comprises a highly conserved central domain forming two antiparallel alpha-helices flanked by the less conserved N- and C-terminal regions forming a five-stranded beta-sandwich. Amino acids from the two helices participate in the generation of a hydrophobic surface area which is conserved in all eukaryotic and archaeal homologous subunits, and likely constitutes a critical macromolecular interaction interface. The hRPABC14.4 structure accounts for mutagenesis results in Saccharomyces cerevisiae and provides a structural working model for elucidating the role of this subunit in the molecular architecture and function of the human nuclear RNA polymerases.
rho-Independent transcription terminators in Escherichia coli contain a dG+dC-rich dyad-symmetrical structure that encodes an RNA hairpin structure and an adjacent, downstream dA+dT-rich region which encodes uridines at the 3'-end of the transcript. In the threonine (thr) attenuator, there are at least six sequence segments in the DNA that might affect termination: the sequence upstream of the attenuator, the deoxythymidine-rich stretch immediately preceding the G+C-rich region, the G+C-rich region itself and its hairpin loop-encoding region, the deoxyadenosine tract following the G+C-rich region, and the following downstream sequence. Our previous studies (Jeng, S.-T., Gardner, J.F., and Gumport, R.I. (1990) J. Biol. Chem. 265, 3823-3830) indicate that both the stability and sequence of the RNA hairpin formed by the G+C-rich region and the length of the uridine tract encoded by the deoxyadenosine stretch influence the termination of T7 RNA polymerase in vitro. In this report, we demonstrate that the template deoxythymidine run upstream of the G+C-rich region, the loop-encoding segment, and the sequences upstream and downstream of the thr attenuator also affect termination. These results indicate that: 1) a deoxythymidine tract is not absolutely required for termination, but increasing the number of deoxythymidines from one to nine base pairs causes T7 RNA polymerase to terminate more efficiently; 2) a template with the natural loop sequence reversed results in a higher termination efficiency than one encoded by the the wild-type attenuator; 3) the termination of T7 RNA polymerase is affected by sequences both proximal and distal to the thr attenuator.
The structure of the elongation complex of vaccinia RNA polymerase halted at discrete template positions was examined by DNase I footprinting. The leading edge of the footprint bore a constant relationship to the catalytic template position, being 22-24 nucleotides (nt) in advance on the nontemplate strand and 17 nt on the template strand. DNase hypersensitivity of the nontemplate strand at the leading edge suggested that the DNA might be distorted as it entered the polymerase molecule. The region of DNA unwinding at the transcription bubble extended at least 12 nt 5' from the catalytic center, as indicated by the reactivity of adenosine residues to diethylpyrocarbonate. Cu-phenanthroline-hypersensitive sites located 13 nt 5' and 4 nt 3' of the growing point appeared to demarcate the margins of the bubble. Strand asymmetry of chemical modification within the bubble was consistent with an RNA-DNA hybrid of no more than 10 base pairs.
The baculovirus Autographa californica nuclear polyhedrosis virus encodes a DNA-dependent RNA polymerase that transcribes viral late genes. This polymerase is composed of four equimolar subunits, LEF-4, LEF-8, LEF-9, and p47. Here we present data indicating that the LEF-4 subunit of RNA polymerase is a guanylyltransferase. Incubation of RNA polymerase in the presence of divalent cation and radiolabeled GTP resulted in the formation of a covalent enzyme-guanylate complex that comigrated with the LEF-4 subunit. The label transfer assay showed an absolute requirement for divalent cation which could be satisfied by either manganese or magnesium. The reaction was specific for guanine nucleotides, and GTP was more effective than dGTP in the formation of enzyme-guanylate complex. To demonstrate that LEF-4 was the guanylyltransferase, the single subunit was overexpressed in baculovirus-infected cells. The overexpressed protein was primarily cytosolic, indicating that other proteins in the RNA polymerase complex were responsible for nuclear targeting of LEF-4. LEF-4 alone was able to covalently bind GMP, although less efficiently than viral RNA polymerase.
The recognition of promoter region -10 nucleotide sequences in prokaryotes is believed to be mediated by a segment of alpha-helix in a region of RNA polymerase sigma factors called 2.4. Earlier genetic studies implicated Thr-100 in region 2.4 of the Bacillus subtilis sigma factor sigma H in the recognition of the G.C base pair at position -13 in the -10 region (GAAT) of a cognate promoter. In confirmation of this assignment, we now show that a change-of-specificity mutant of sigma H in which Thr-100 was replaced with isoleucine suppresses a G.C----A.T nucleotide substitution at position -13 but not other "promoter down mutations" (causing impaired promoter activity) at positions -13, -12, and -11. We also show that a loss-of-contact mutant created by the replacement of Thr-100 with alanine (having a short side chain) enables sigma H to tolerate three different promoter down mutations at position -13 but not down mutations at other positions. Finally, we suggest the identification of an additional amino acid involved in base-pair recognition by the demonstration that the replacement of Arg-96 with alanine specifically suppresses an A.T----G.C promoter down mutation at position -12. The identification of amino acids that are four residues apart that are involved in the recognition of adjacent base pairs may fix the orientation of region 2.4 (its NH2 terminus being proximal to the promoter transcription start site) and is consistent with a model in which the recognition of promoter region -10 nucleotide sequences is mediated by an alpha-helix in which residues involved in base-pair contact are separated by one turn and clustered on one face of the helix.
Bacteriophage T7 RNA polymerase (T7 RNAP) is known to be one of the simplest enzymes catalyzing RNA synthesis. In contrast to most RNA polymerases known, this enzyme consists of one subunit and is able to carry out transcription in the absence of additional protein factors. Owing to its molecular properties, the enzyme is widely used for synthesis of specific transcripts, as well as being a suitable model for studying the mechanisms of transcription. In this minireview the recent data on the structure and mechanism of T7 RNAP, including enzyme-promoter interactions, principal stages of transcription, and the results of functional studies are discussed.
Various Escherichia coli promoters contain, in addition to the classical -35 and -10 hexamers, a third recognition element, named the UP element. Located upstream of the -35 box, UP elements stimulate promoter activity by forming a docking site for the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). Accumulating genetic, biochemical and structural information has provided a detailed picture on the molecular mechanism underlying UP element-dependent promoter stimulation in E.coli. However, far less is known about functional UP elements of Bacillus subtilis promoters. Here we analyse the strong early sigma(A)-RNA polymerase-dependent promoters C2, A2c and A2b of the lytic B.subtilis phage phi29. We demonstrate that the phage promoters contain functional UP elements although their contribution to promoter strength is very different. Moreover, we show that the UP element of the A2b promoter, being critical for its activity, is located further upstream of the -35 box than most E.coli UP elements. The importance of the UP elements for the phage promoters and how they relate to other UP elements are discussed.
Current requirement for description of each new promoter assumes identification of all DNA-protein and protein-protein contacts important for transcription complex formation. Experimental approaches allow estimating which one of seven alternative sigma-subunits is employed for RNA synthesis and verifying transcription dependence on known regulatory proteins. Promoter sequence by itself also contains this information. That is why, the type of promoter as well as potential regulatory proteins with high probability may be proposed, if the transcription start point has been determined. Transcription activity of the promoter is usually less predictive. It depends on the specific contacts formed by sigma-subunits with correspondent conservative elements and on many other non-specific factors that are hardly taken into account. Interaction with RNA polymerase alpha-subunits seems does not require any particular functional group of nucleotides thus exemplifying non-sequence-specific binding within binary polymerase-promoter complexes. The role of this interaction in the transcription complex formation is the main subject of this survey that summarizes our own experimental results and the data of other authors. Attempts have been made to compare nucleotide sequences of the promoters recognized by different sigma-factors within putative contact regions with alpha-subunits and to discuss regulatory propensity of free alpha-subunits.
The C-terminal domain of the alpha subunit (alpha CTD) of Escherichia coli RNA polymerase is often involved in transcriptional regulation. The alpha CTD typically stimulates transcription via interactions with promoter UP element DNA and transcriptional activators. DNase I footprinting and gel mobility shift assays were used to look for potential interaction of the alpha CTD with the phage Mu middle promoter P(m) and its activator protein Mor. Binding of RNA polymerase to P(m) in the presence of Mor resulted in production of a DNase I footprint downstream of Mor due to open complex formation and generation of a second footprint just upstream of the Mor binding site. Generation of the upstream footprint did not require open complex formation and also occurred in reactions in which the alpha CTD or His-alpha proteins were substituted for RNA polymerase. In gel mobility shift assays, the formation of a supershifted ternary complex demonstrated that Mor and His-alpha bind synergistically to P(m) DNA. Gel shift assays with short DNA fragments demonstrated that only the Mor binding site and a single upstream alpha CTD binding site were required for ternary complex formation. These results suggest that the alpha CTD plays a role in P(m) transcription by binding to P(m) DNA just upstream from Mor and making protein-protein interactions with Mor that stabilize the binding of both proteins.
The red clover necrotic mosaic virus (RCNMV) genome is split between two single-stranded RNA species termed RNA-1 and RNA-2. RNA-1 directs the synthesis of 88-kDa (p88), 57-kDa (p57), 37-kDa (p37), and 27-kDa (p27) polypeptides and RNA-2 a 35-kDa (p35) polypeptide in vitro. The coding order of the RNA-1 products was determined to be 5'-p27-p57-p37-3'. Antibodies to synthetic peptides representing the carboxyl terminal portions of p27 and p57 immunoprecipitated their respective polypeptides in addition to p88, suggesting that p88 is a fusion protein. A frameshift heptanucleotide sequence element has been identified in RCNMV RNA-1. In addition, a stable stem-loop secondary structure adjacent to the heptanucleotide sequence is predicted. Together, these sequence elements suggest that a ribosomal frameshifting event occurs which allows translational readthrough of the p27 open reading frame into the p57 open reading frame, generating the observed p88 product. An RNA-1 expression construct fusing the p57 and the CP open reading frame was engineered to investigate the ribosomal frameshifting event. CP antibodies immunoprecipitated a fusion protein of the predicted size containing the carboxyl portion of CP. Site-directed mutagenesis of the frameshift element indicates that in vitro, p88 can also be expressed alternatively by suppression of an amber termination codon. Based on these data, we propose that the putative RCNMV RNA polymerase is an 88-kDa polypeptide expressed by a ribosomal frameshifting mechanism similar to those utilized by retroviruses.
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The phage T7 and SP6 RNA polymerase-promoter systems are very similar in many characteristics, but maintains stringent specificity for each. In order to identify the base pair element that distinguishes between T7 and SP6 promoters, the base pairs at -12, -10, -9, and -8 of the T7 promoter consensus sequence were changed singly and multiply to the SP6 promoter-specific base pairs, and assayed for T7 and SP6 promoter activities. The results indicate that the primary discrimination element is the base pairs at -8 and -9. The two-base-pair substitution alone in T7 promoter by SP6-specific base pairs is sufficient to make the T7 variant be a SP6 promoter, abolishing T7 promoter activity.
A set of 18 variant lac UV5 promoters was constructed, each carrying a single base substitution within the -35 region (nucleotide positions from -36 to -31 relative to the transcription start site). Using truncated DNA fragments carrying these variant promoters and purified Escherichia coli RNA polymerase holoenzyme, in vitro mixed transcription assays were performed to determine two parameters governing promoter strength: i.e., the binding affinity to RNA polymerase (parameter I) and the rate of open complex formation (parameter II). The following conclusions were drawn from the data presented: (1) Alteration in the promoter strength of variant promoters is dependent on both the position and base species of substitutions; (2) the consensus sequence (TTGACA) exhibits the highest values for both parameters; (3) base substitutions at nucleotide position -34 cause marked effect on both parameters; (4) cytosine at nucleotide position -32 can not be replaced with other nucleotides without significant reduction of the promoter strength; and (5) base substitution at nucleotide position -31 exerts only a little effect on parameter I. All these findings were confirmed by abortive initiation assays.
Transcriptional pausing by RNA polymerase (RNAP) plays an important role in the regulation of gene expression. Defined, sequence-specific pause sites have been identified biochemically. Single-molecule studies have also shown that bacterial RNAP pauses frequently during transcriptional elongation, but the relationship of these "ubiquitous" pauses to the underlying DNA sequence has been uncertain. We employed an ultrastable optical-trapping assay to follow the motion of individual molecules of RNAP transcribing templates engineered with repeated sequences carrying imbedded, sequence-specific pause sites of known regulatory function. Both the known and ubiquitous pauses appeared at reproducible locations, identified with base-pair accuracy. Ubiquitous pauses were associated with DNA sequences that show similarities to regulatory pause sequences. Data obtained for the lifetimes and efficiencies of pauses support a model where the transition to pausing branches off of the normal elongation pathway and is mediated by a common elemental state, which corresponds to the ubiquitous pause.
In chloroplasts, the 3' untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3'-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3' IR-containing RNA (3' IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b6/f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T1 digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3' IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3' untranslated region, only a single copy, which we have termed box II, appears to be essential for in vitro protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3'-end processing and/or influence the stability of petD mRNA in chloroplasts.
We have used photocrosslinking to analyze the contacts between the 3' end of the RNA and Escherichia coli RNA polymerase during the early steps of RNA synthesis using the nucleotide analog 8-azido-ATP (8-N3-ATP). The crosslinking group on 8-N3-ATP contacts the beta, beta' and sigma subunits when the analog is bound to the holoenzyme. We show here that 8-N3-ATP is a substrate for E. coli RNA polymerase and acts as an RNA chain terminator when incorporated into the 3' end of nascent RNA. 8-N3-AMP was incorporated uniquely at the 3' end of tri-, tetra- and pentanucleotides synthesized from a poly[d(A-T)] template and at the 3' end of pentanucleotides from two promoters (lambda PR' and E. coli rrnB P1). The oligonucleotides were covalently attached to the RNA polymerase by irradiation of transcription complexes with ultraviolet light. All RNAs labeled the beta and beta' subunits, but sigma was contacted only by the trinucleotide and tetranucleotide on poly[d(A-T)]. Sigma is still present in transcription complexes containing the pentanucleotide on poly[d(A-T)], despite the lack of labeling. Neither pentanucleotide from the authentic promoters contacted sigma. We conclude that as holoenzyme moves downstream, either two separate conformational changes occur, after synthesis of the trinucleotide and tetranucleotide, which result in movement of sigma away from the nucleotide binding site or, alternatively, sigma remains fixed relative to the DNA while the domain on core polymerase forming the nucleotide binding site moves downstream.
We affinity-purified the tobacco plastid-encoded plastid RNA polymerase (PEP) complex by the alpha subunit containing a C-terminal 12 x histidine tag using heparin and Ni(2+) chromatography. The composition of the complex was determined by mass spectrometry after separating the proteins of the >900 kDa complex in blue native and SDS polyacrylamide gels. The purified PEP contained the core alpha, beta, beta', beta" subunits and five major associated proteins of unknown function, but lacked sigma factors required for promoter recognition. The holoenzyme efficiently recognized a plastid psbA promoter when it was reconstituted from the purified PEP and recombinant plastid sigma factors. Reconstitution of a plastid holoenzyme with individual sigma factors will facilitate identification of sigma factor-specific promoter elements.