The RNA polymerase III transcription apparatus.
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Animals↗
Biomedical subjects
E P Geiduschek
Publications and source records attributed to E P Geiduschek.
The RNA polymerase III transcription initiation factor TFIIIB participates in two steps of promoter opening.
Evidence for post-recruitment functions of yeast transcription factor (TF)IIIB in initiation of transcription was first provided by the properties of TFIIIB-RNA polymerase III-promoter complexes assembled with deletion mutants of its Brf and B" subunits that are transcriptionally inactive because they fail to open the promoter. The experiments presented here show that these defects can be repaired by unpairing short (3 or 5 bp) DNA segments spanning the transcription bubble of the open promoter complex. Analysis of this suppression phenomenon indicates that TFIIIB participates in two steps of promoter opening by RNA polymerase III that are comparable to the successive steps of promoter opening by bacterial RNA polymerase holoenzyme. B" deletions between amino acids 355 and 421 interfere with the initiating step of DNA strand separation at the upstream end of the transcription bubble. Removing an N-terminal domain of Brf interferes with downstream propagation of the transcription bubble to and beyond the transcriptional start site.
Base Sequence↗
A thermostable platform for transcriptional regulation: the DNA-binding properties of two Lrp homologs from the hyperthermophilic archaeon Methanococcus jannaschii.
The hyperthermophilic archaeon Methanococcus jannaschii encodes two putative transcription regulators, Ptr1 and Ptr2, related to the bacterial Lrp/AsnC family of transcriptional regulators. We show that these two small helix-turn-helix proteins are specific DNA-binding proteins recognizing sites in their respective promoter regions. In vitro selection at high temperature has been used to isolate sets of high- affinity DNA sites that define a palindromic consensus binding sequence for each protein. Ptr1 and Ptr2 bind these cognate sites from one side of the DNA helix, as dimers, with each protein monomer making base- specific contacts in the major groove. As the first archaeal DNA-binding proteins with clearly defined specificities, Ptr1 and Ptr2 provide a thermostable DNA-binding platform for analysis of effector interactions with the core archaeal transcription apparatus; a platform allowing manipulation of promoter structure and examination of mechanisms of action at heterologous promoters.
Amino Acid Sequence↗
Solution structure of a mutant of transcription factor 1: implications for enhanced DNA binding.
An NMR solution structure of a mutant of the homodimer protein transcription factor 1, TF1-G15/I32 (22 kDa), has been solved to atomic resolution, with 23 final structures that converge to an r.m. s.d. of 0.78 A. The overall shape of TF1-G15/I32 remains similar to that of the wild-type protein and other type II DNA-binding proteins. Each monomer has two N-terminal alpha-helices separated by a short loop, followed by a three-stranded beta-sheet, whose extension between the second and third beta-strands forms an antiparallel beta-ribbon arm, leading to a C-terminal third alpha-helix that is severely kinked in the middle. Close examination of the structure of TF1-G15/I32 reveals why it is more stable and binds DNA more tightly than does its wild-type counterpart. The dimeric core, consisting of the N-terminal helices and the beta-sheets, is more tightly packed, and this might be responsible for its increased thermal stability. The DNA-binding domain, composed of the top face of the beta-sheet, the beta-ribbon arms and the C-terminal helices, is little changed from wild-type TF1. Rather, the enhancement in DNA affinity must be due almost exclusively to the creation of an additional DNA-binding site at the side of the dimer by changes affecting helices 1 and 2: helix 2 of TF1-G15/I32 is one residue longer than helix 2 of the wild-type protein, bends inward, and is both translationally and rotationally displaced relative to helix 1. This rearrangement creates a longer, narrower fissure between the V-shaped N-terminal helices and exposes additional positively charged surface at each side of the dimer.
Bacillus subtilis↗
The Brf and TATA-binding protein subunits of the RNA polymerase III transcription factor IIIB mediate position-specific integration of the gypsy-like element, Ty3.
Ty3 integrates into the transcription initiation sites of genes transcribed by RNA polymerase III. It is known that transcription factors (TF) IIIB and IIIC are important for recruiting Ty3 to its sites of integration upstream of tRNA genes, but that RNA polymerase III is not required. In order to investigate the respective roles of TFIIIB and TFIIIC, we have developed an in vitro integration assay in which Ty3 is targeted to the U6 small nuclear RNA gene, SNR6. Because TFIIIB can bind to the TATA box upstream of the U6 gene through contacts mediated by TATA-binding protein (TBP), TFIIIC is dispensable for in vitro transcription. Thus, this system offers an opportunity to test the role of TFIIIB independent of a requirement of TFIIIC. We demonstrate that the recombinant Brf and TBP subunits of TFIIIB, which interact over the SNR6 TATA box, direct integration at the SNR6 transcription initiation site in the absence of detectable TFIIIC or TFIIIB subunit B". These findings suggest that the minimal requirements for pol III transcription and Ty3 integration are very similar.
Bacterial Proteins↗
Mechanisms for the enhanced thermal stability of a mutant of transcription factor 1 as explained by (1)H, (15)N and (13)C NMR chemical shifts and secondary structure analysis.
A variant of the bacteriophage SPO1-encoded transcription factor 1 (TF1) with two site-specific mutations (E15G and T32I) was shown to be more thermally stable and bind DNA more tightly compared to the wild-type protein. In order to understand the biochemical mechanisms underlying these properties, we are engaged in determining the solution structures of this mutant alone and in complex with DNA using nuclear magnetic resonance (NMR) spectroscopy. The first phase of this project is reported here, as we have completed most of the backbone and sidechain sequential NMR assignments of the mutant protein, TF1-G15/I32. Insights derived from the (1)H, (15)N and (13)C chemical shifts and from the secondary structure analysis provide us with an explanation for the noted increase in thermal stability of TF1-G15/I32. Compared to the structure of the wild-type protein, the beta-sheet and the C-terminal helix remain largely unaffected whereas the mutations cause great changes in the first two helices and their enclosed loop. Specifically, we have found that the second helix is extended by one residue at its N-terminus and rotated in a way that allows Ala-37 to interact with Tyr-94 of the C-terminal helix. The loop has been found to become more rigid as a result of hydrophobic interactions between the flanking second and first helices and also between the second helix and the loop itself. Furthermore, the T32I mutation allows tighter packing between the second helix and the beta-sheet. Collectively, these changes contribute to a more tightly associated dimer and hence, to a greater thermal stability.
DNA-Binding Proteins↗
The orientation of DNA in an archaeal transcription initiation complex.
RNA polymerase from the hyperthermophile archaeon Pyrococcus furiosus (Pfu) forms specific and transcriptionally active complexes with its conjugate transcription factors TBP (the archaeal TATA binding protein homolog) and TFB (the archaeal homolog of eukaryotic RNA polymerase II and III transcription factors TFIIB and Brf) at the Pfu glutamate dehydrogenase promoter. A photochemical crosslinking method was used to map the vicinity of the catalytic subunits of Pfu RNA polymerase to DNA locations distributed along the polymerase-promoter interface. The largest component of this archaeal polymerase is split into two subunits, A' and A", whose relatively sharp boundary of DNA crosslinking (probed on the transcribed strand) is centered five to six base pairs downstream of the transcriptional start site. A strong argument based on this information, on the well-defined homology between the core bacterial, archaeal and eukaryotic RNA polymerase subunits, and on the recently determined structure of a bacterial RNA polymerase specifies the directionality of DNA in the archaeal transcription complex and its trajectory downstream of the transcriptional start site.
Archaeal Proteins↗
Alignment of the B" subunit of RNA polymerase III transcription factor IIIB in its promoter complex.
TFIIIB, the central transcription initiation factor of the eukaryotic nuclear RNA polymerase (pol) III is composed of three subunits: the TATA-binding protein; Brf, the TFIIB-related subunit; and B", the Saccharomyces cerevisiae, TFC5 gene product. The orientation of the B" subunit within the TFIIIB-DNA complex has been analyzed at two promoters by two approaches that involve site-specific photochemical protein-DNA cross-linking: a collection of B" internal and external deletion proteins has been surveyed for those deletions that alter the interaction of B" with DNA or change the orientation of B" relative to DNA; a method for regionally mapping cross-links between specific DNA sites and (32)P-end-labeled protein has also been applied. The results map an N-proximal segment of B" to the upstream end of the TFIIIB-DNA complex and amino acids 299-315 to the principal DNA-contact site, approximately 8 base pairs upstream of the TATA box. The analysis also indicates that a segment comprising amino acids 316-434 loops away from DNA, and locates the C-proximal 170 amino acids of B" downstream of the TATA box. Examination of two-cross-link products formed by DNA with adjacent and nearby photoactive nucleotides supports the conclusion that Brf and B" share an extended interface along the length of the TFIIIB-DNA complex.
Base Sequence↗
A minimal RNA polymerase III transcription system.
Transcription factor (TF) IIIB recruits RNA polymerase (pol) III for specific initiation of transcription. All three subunits of TFIIIB, TBP, Brf (the TFIIB-related subunit) and B", are required for transcription of supercoiled and linear duplex DNA, but we show here that B" is non-essential on a promoter that has been partly pre-opened by unpairing a short segment of the transcription bubble. These findings expose a striking similarity between transcriptional initiation by pol II, pol III and bacterial RNA polymerases: a preformed single-stranded DNA bubble upstream of the transcriptional start removes the dependence of pol II on TFIIE, TFIIH and ATP hydrolysis, and the dependence of pol III on B"; the favored placement of the transcription bubble for B"-independent transcription by pol III overlaps a DNA segment that interacts sequence specifically as single-stranded DNA with the sigma(70 )initiation subunit of Escherichia coli RNA polymerase holoenzyme.
Adenosine Triphosphate↗
Sigma competition: the contest between bacteriophage T4 middle and late transcription.
In bacterial transcription, diverse sigma-family promoter recognition proteins compete for a common RNA polymerase core. Bacteriophage T4 infection ultimately reduces this competition to a duel between activated viral middle and enhanced late transcription, involving two sigma proteins, two phage-encoded activator proteins and two phage-specific co-activators. This competition has been analyzed in vitro, and the relative abundances in T4-infected Escherichia coli of the participating proteins have been measured. Activated late transcription holds an advantage over activated middle transcription, especially at higher ionic strength. This advantage is further compounded by ADP-ribosylation of the RNA polymerase alpha subunits, and by the phage-specific, RNA polymerase core-bound RpbA subunit. The largest contribution to the middle-late competition is made by gp55, the late sigma factor, but not enough of gp55 is produced during T4 infection to shut off middle transcription by direct competition with sigma(70). AsiA, the originally identified anti-sigma protein is not a major determinant of middle-late competition.
Animals↗
The RNA polymerase III-recruiting factor TFIIIB induces a DNA bend between the TATA box and the transcriptional start site.
TFIIIB, the RNA polymerase III-recruiting factor of Saccharomyces cerevisiae, may be assembled upstream of the transcriptional start site, either through the interaction of its constituent TATA-binding protein (TBP) with a strong TATA-box, or by means of the multisubunit assembly factor, TFIIIC. Missing nucleoside interference analysis of TFIIIC-dependent TFIIIB-DNA complex formation revealed enhanced complex formation at 0 degreesC when the DNA is missing nucleosides in two broad 7-10 bp regions centered around base-pairs -17 and -3 relative to the transcriptional start site; no effect of missing nucleosides was evident at 20 degreesC. The implication of these results for required DNA flexure in TFIIIC-mediated TFIIIB-DNA complex formation was pursued in a TFIIIC-independent context, using DNA with a suboptimal 6 bp TATA box (TATAAA). A unique missing nucleoside at the downstream end of the TATA box, corresponding to the position of one of two TBP-mediated DNA kinks, significantly enhances TBP-DNA complex formation. In contrast, TFIIIB displays a broad preference for missing nucleosides within an approximately 15 bp region immediately downstream of the TATA box. Consecutive mismatches (4-nt loops), either at the sites of TBP-mediated DNA kinking at both ends of the TATA box or within the identified region where missing nucleosides promote TFIIIB-DNA complex formation, also result in enhanced and specific TFIIIB assembly; 4-nt loops further downstream do not lead to preferential placement of TFIIIB. We conclude that TFIIIB induces an additional DNA deformation between the TATA box and the start site of transcription that is likely to be more extended than the sharp kinks generated by TBP.
Base Sequence↗
Abortive initiation of transcription at a hybrid promoter. An analysis of the sliding clamp activator of bacteriophage T4 late transcription, and a comparison of the sigma70 and T4 gp55 promoter recognition proteins.
Bacteriophage T4 late promoters are transcribed by an RNA polymerase holoenzyme comprising the Escherichia coli core, E, the phage gene 55-encoded promoter recognition subunit, gp55, and the gene 33-encoded co-activator, gp33. Transcriptional initiation is activated by the T4 gene 45-encoded sliding clamp, which is loaded on to DNA at enhancer-like sites by its clamp-loader. Correct initiation of transcription at late promoters in basal mode requires only RNA polymerase core and gp55 (E.gp55). Dinucleotide-primed abortive initiation of basal and activated T4 late transcription has been compared. Only the trinucleotide non-productive transcript is made at a high rate; all other short transcripts are made at rates of less than one molecule per productive transcript. Gp45 increases abortive trinucleotide synthesis along with productive transcription, although the proportion of productive transcripts is also elevated. Nevertheless, this increase accounts for only a small part of the activation of T4 late transcription that is generated by its activator and co-activator. The pattern of production of short transcripts differs subtly between basal and enhanced transcription, indicating that linking the RNA polymerase with its sliding clamp activator only generates minor changes in the transition from abortive to productive RNA chain elongation. The T4 late promoter is converted to a strong sigma70 promoter by inserting an appropriate -35 promoter element. A direct comparison at such a hybrid promoter shows sigma70 and gp55 generating qualitatively and quantitative different patterns of abortive initiation at the same start site.
Bacteriophage T4↗
Activator-sigma interaction: A hydrophobic segment mediates the interaction of a sigma family promoter recognition protein with a sliding clamp transcription activator.
Activation of transcription at bacteriophage T4 late promoters and coupling of late transcription to concurrent replication requires a peculiar transcriptional activator, the gp45 sliding clamp of the T4 DNA polymerase. In order to activate transcription, the topologically DNA-linked trimeric gp45 must interact with two T4-encoded RNA polymerase-binding proteins, the gp33 co-activator, and the gp55 late sigma factor. The carboxy termini of gp55 and gp33 share a similar sequence, which has been shown to be required for response of late transcription to activation by gp45. Alanine-scanning mutagenesis of the C terminus of gp55 shows that residues within the short hydrophobic sequence L(D/A)FLYE, are necessary for gp55 to bind to gp45, and to respond maximally to transcriptional activation by gp45. When fused to GST, the peptide SLDFLYE suffices for specific gp45 binding. Thus, it constitutes the main gp55 epitope for gp45 interaction.
Alanine↗
Affinity, stability and polarity of binding of the TATA binding protein governed by flexure at the TATA Box.
The TATA binding protein (TBP), which plays a central role in gene regulation as an essential component of all three nuclear transcription systems, sharply kinks the TATA box at two sites and severely contorts the intervening DNA segment. DNA constructs with precisely localized flexure have been used to investigate the special repertoire of mechanisms and properties that arise from TBP interacting with the TATA box. DNA flexure precisely localized to the sites of TBP-mediated DNA kinking increases the affinity of TBP more than 100-fold; unexpectedly, this increase in affinity is achieved almost exclusively by increasing the stability of the TBP-DNA complex rather than the rate of its formation. In vitro transcription with RNA polymerase III provides a first demonstration that the orientation of TBP on the TATA box is governed by DNA deformability, its C-proximal repeat contacting the more flexible end of the TATA box. Exceptionally stable TBP-DNA complexes reach their orientational equilibrium very slowly; in these circumstances, assembly of stable ("committed") transcription initiation complexes can freeze far-from-equilibrium orientations of TBP on the TATA box, causing transcription polarity to be determined by a kinetic trapping mechanism.
Base Pairing↗
A post-recruitment function for the RNA polymerase III transcription-initiation factor IIIB.
Transcription factor (TF) IIIB, which directs RNA polymerase (pol) III to its promoters, is made up of three components: the TATA box-binding protein, the TFIIB-related Brf, and the pol III-specific B". Certain mutations in Saccharomyces cerevisiae Brf and B" retain TFIIIB transcription factor activity with supercoiled DNA but are inactive with linear duplex DNA. Further analysis shows that these inactive TFIIIB-DNA complexes bind pol III and position it appropriately over the transcriptional start site but do not form DNA strand-separated open promoter complexes. It is proposed that the normal function of TFIIIB combines pol III recruitment with an active role in a subsequent step of transcriptional initiation leading to promoter opening.
Base Sequence↗
Chromatin transcription: clearing the gridlock.
A factor has been purified that specifically facilitates RNA chain elongation in a chromatin context; the properties of this factor help to define the challenges that lie ahead in understanding how RNA polymerase manages to transcribe DNA that is packaged into chromatin.
Chromatin↗