Transcription Factor B Contacts Promoter DNA Near the Transcription Start Site of the Archaeal Transcription Initiation Complex

In standard transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA, selects a transcription start site, and--using a "scrunching" mechanism, in which RNAP remains bound to the promoter, unwinds additional DNA, and pulls the additional unwound DNA past its active center, synthesizing an RNA product having a 5' sequence complementary to the DNA template. In an alternative pathway of transcription initiation, termed "reiterative transcription initiation," primarily observed at promoters containing homopolymeric sequences at or near the transcription start site, RNAP binds to promoter DNA, unwinds promoter DNA, selects a transcription start site, and--using a mechanism that has not previously been defined--generates an RNA product having a 5' sequence that contains a variable number of nucleotides not complementary to the DNA template. Here, using x-ray crystallography to define structures, using protein-DNA-photocrosslinking to map positions of RNAP leading and trailing edges relative to DNA, and using single-molecule DNA nanomanipulation to assess RNAP-dependent DNA unwinding, we show that RNA extension in reiterative transcription initiation (1) occurs without DNA scrunching, (2) involves a short, 2 bp (post-translocated state) to 3 bp (pre-translocated state) RNA-DNA hybrid, (3) and can involve an RNA product positioned as in standard transcription initiation and a DNA template strand positioned differently from standard transcription initiation. The results establish that, whereas RNA extension in standard transcription initiation proceeds through a scrunching mechanism, RNA extension in reiterative transcription initiation proceeds through a slippage mechanism, with sliding of RNA relative to DNA within a short, 2-3 bp, RNA-DNA hybrid.

Download Full-text

The mechanism of variability in transcription start site selection

eLife ◽

10.7554/elife.32038 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 4

Author(s):

Libing Yu ◽

Jared T Winkelman ◽

Chirangini Pukhrambam ◽

Terence R Strick ◽

Bryce E Nickels ◽

...

Keyword(s):

Transcription Start Site ◽

Single Molecule ◽

Transcription Initiation ◽

General Mechanism ◽

Start Site ◽

Transcription Start ◽

Transcription Bubble ◽

Promoter Dna

During transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA to form an RNAP-promoter open complex (RPo) containing a single-stranded ‘transcription bubble,’ and selects a transcription start site (TSS). TSS selection occurs at different positions within the promoter region, depending on promoter sequence and initiating-substrate concentration. Variability in TSS selection has been proposed to involve DNA ‘scrunching’ and ‘anti-scrunching,’ the hallmarks of which are: (i) forward and reverse movement of the RNAP leading edge, but not trailing edge, relative to DNA, and (ii) expansion and contraction of the transcription bubble. Here, using in vitro and in vivo protein-DNA photocrosslinking and single-molecule nanomanipulation, we show bacterial TSS selection exhibits both hallmarks of scrunching and anti-scrunching, and we define energetics of scrunching and anti-scrunching. The results establish the mechanism of TSS selection by bacterial RNAP and suggest a general mechanism for TSS selection by bacterial, archaeal, and eukaryotic RNAP.

Download Full-text

The mechanism of transcription start site selection

10.1101/156877 ◽

2017 ◽

Author(s):

Libing Yu ◽

Jared T. Winkelman ◽

Chirangini Pukhrambam ◽

Terence R. Strick ◽

Bryce E. Nickels ◽

...

Keyword(s):

Transcription Start Site ◽

Single Molecule ◽

Transcription Initiation ◽

General Mechanism ◽

Start Site ◽

Transcription Start ◽

Transcription Bubble ◽

Promoter Dna

SUMMARYDuring transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA to form an RNAP-promoter open complex (RPo) containing a single-stranded "transcription bubble," and selects a transcription start site (TSS). TSS selection occurs at different positions within the promoter region, depending on promoter sequence and initiating-substrate concentration. Variability in TSS selection has been proposed to involve DNA "scrunching" and "antiscrunching," the hallmarks of which are: (i) forward and reverse movement of the RNAP leading edge, but not trailing edge, relative to DNA, and (ii) expansion and contraction of the transcription bubble. Here, using in vitro and in vivo protein-DNA photocrosslinking and single-molecule nanomanipulation, we show bacterial TSS selection exhibits both hallmarks of scrunching and anti-scrunching, and we define energetics of scrunching and anti-scrunching. The results establish the mechanism of TSS selection by bacterial RNAP and suggest a general mechanism for TSS selection by bacterial, archaeal, and eukaryotic RNAP.

Download Full-text

Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1603271113 ◽

2016 ◽

Vol 113 (21) ◽

pp. E2899-E2905 ◽

Cited By ~ 23

Author(s):

Irina O. Vvedenskaya ◽

Hanif Vahedian-Movahed ◽

Yuanchao Zhang ◽

Deanne M. Taylor ◽

Richard H. Ebright ◽

...

Keyword(s):

Rna Polymerase ◽

Transcription Start Site ◽

Transcription Initiation ◽

Specific Protein ◽

Start Site ◽

Transcription Start ◽

Transcription Bubble ◽

Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

Download Full-text

Transcription Regulation by Tandem-Bound FNR at Escherichia coli Promoters

Journal of Bacteriology ◽

10.1128/jb.185.20.5993-6004.2003 ◽

2003 ◽

Vol 185 (20) ◽

pp. 5993-6004 ◽

Cited By ~ 17

Author(s):

Anne M. L. Barnard ◽

Jeffrey Green ◽

Stephen J. W. Busby

Keyword(s):

Escherichia Coli ◽

Transcription Factor ◽

Binding Site ◽

Transcription Regulation ◽

Transcription Start Site ◽

Promoter Activity ◽

Start Site ◽

Transcription Start ◽

Substitution Mutant

ABSTRACT FNR is an Escherichia coli transcription factor that regulates the transcription of many genes in response to anaerobiosis. We have constructed a series of artificial FNR-dependent promoters, based on the melR promoter, in which a consensus FNR binding site was centered at position −41.5 relative to the transcription start site. A second consensus FNR binding site was introduced at different upstream locations, and promoter activity was assayed in vivo. FNR can activate transcription from these promoters when the upstream FNR binding site is located at many different positions. However, sharp repression is observed when the upstream-bound FNR is located near positions −85 or −95. This repression is relieved by the FNR G74C substitution mutant, previously identified as being defective in transcription repression at the yfiD promoter. A parallel series of artificial FNR-dependent promoters, carrying a consensus FNR binding site at position −61.5 and a second upstream DNA site for FNR, was also constructed. Again, promoter activity was repressed by FNR when the upstream-bound FNR was located at particular positions.

Download Full-text

Core Promoter-Dependent TFIIB Conformation and a Role for TFIIB Conformation in Transcription Start Site Selection

Molecular and Cellular Biology ◽

10.1128/mcb.22.19.6697-6705.2002 ◽

2002 ◽

Vol 22 (19) ◽

pp. 6697-6705 ◽

Cited By ~ 24

Author(s):

Jennifer A. Fairley ◽

Rachel Evans ◽

Nicola A. Hawkes ◽

Stefan G. E. Roberts

Keyword(s):

Transcription Start Site ◽

Site Selection ◽

Transcription Initiation ◽

Core Promoter ◽

Initiation Site ◽

Start Site ◽

Wild Type ◽

Transcription Start ◽

General Transcription Factor ◽

Selection Of

ABSTRACT The general transcription factor TFIIB plays a central role in the selection of the transcription initiation site. The mechanisms involved are not clear, however. In this study, we analyze core promoter features that are responsible for the susceptibility to mutations in TFIIB and cause a shift in the transcription start site. We show that TFIIB can modulate both the 5′ and 3′ parameters of transcription start site selection in a manner dependent upon the sequence of the initiator. Mutations in TFIIB that cause aberrant transcription start site selection concentrate in a region that plays a pivotal role in modulating TFIIB conformation. Using epitope-specific antibody probes, we show that a TFIIB mutant that causes aberrant transcription start site selection assembles at the promoter in a conformation different from that for wild-type TFIIB. In addition, we uncover a core promoter-dependent effect on TFIIB conformation and provide evidence for novel sequence-specific TFIIB promoter contacts.

Download Full-text

Role of the Transcription Start Site Core Region and Transcription Factor YY1 in Rous Sarcoma Virus Long Terminal Repeat Promoter Activity

Journal of Virology ◽

10.1128/jvi.72.8.6592-6601.1998 ◽

1998 ◽

Vol 72 (8) ◽

pp. 6592-6601 ◽

Cited By ~ 8

Author(s):

Constance M. Mobley ◽

Linda Sealy

Keyword(s):

Transcription Factor ◽

Transcription Start Site ◽

Rous Sarcoma Virus ◽

Promoter Activity ◽

Transcriptional Activity ◽

Start Site ◽

Transcription Start ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Transcription Factor Yy1

ABSTRACT The Rous sarcoma virus (RSV) long terminal repeat (LTR) contains a transcriptionally potent enhancer and promoter that functions in a variety of cell types. Previous studies have identified the viral sequences required for enhancer activity, and characterization of these elements has provided insight into the mechanism of RSV transcriptional activity. The objective of this study was to better define the RSV LTR promoter by examining the transcription start site core (TSSC) region. Deletion of the TSSC resulted in complete loss of transcriptional activity despite the presence of a functional TATA box, suggesting that the TSSC is required for viral expression. Homologies within the TSSC to the DNA binding motif of YY1 suggested that it might regulate promoter activity. YY1 has been shown to regulate transcription in some cellular genes and viral promoters by binding to sites overlapping the transcription start site. Gel shift assays using YY1 antibody identified YY1 as one of three complexes that bound to the TSSC. Mutation of the YY1 binding site reduced RSV transcriptional activity by more than 50%, suggesting that YY1, in addition to other TSSC-binding factors, regulates RSV transcription. Furthermore, in vitro transcription assays performed with Drosophila embryo extract (devoid of YY1 activity) showed decreased levels of RSV transcription, while transient transfection experiments overexpressing YY1 demonstrated that YY1 could transactivate the RSV LTR ∼6- to 7-fold. We propose that the TSSC plays a vital role in RSV transcription and that this function is partially carried out by the transcription factor YY1.

Download Full-text

Archaeal Transcription: Function of an Alternative Transcription Factor B from Pyrococcus furiosus

Journal of Bacteriology ◽

10.1128/jb.01498-07 ◽

2007 ◽

Vol 190 (1) ◽

pp. 157-167 ◽

Cited By ~ 25

Author(s):

Michael Micorescu ◽

Sebastian Grünberg ◽

Andreas Franke ◽

Patrick Cramer ◽

Michael Thomm ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Initiation ◽

Pyrococcus Furiosus ◽

Factor B ◽

Transcription Bubble ◽

Alternative Transcription ◽

N Terminus ◽

Archaeal Transcription ◽

Transcription Complexes ◽

Low Activity

ABSTRACT The genome of the hyperthermophile archaeon Pyrococcus furiosus encodes two transcription factor B (TFB) paralogs, one of which (TFB1) was previously characterized in transcription initiation. The second TFB (TFB2) is unusual in that it lacks recognizable homology to the archaeal TFB/eukaryotic TFIIB B-finger motif. TFB2 functions poorly in promoter-dependent transcription initiation, but photochemical cross-linking experiments indicated that the orientation and occupancy of transcription complexes formed with TFB2 at the strong gdh promoter are similar to the orientation and occupancy of transcription complexes formed with TFB1. Initiation complexes formed by TFB2 display a promoter opening defect that can be bypassed with a preformed transcription bubble, suggesting a mechanism to explain the low TFB2 transcription activity. Domain swaps between TFB1 and TFB2 showed that the low activity of TFB2 is determined mainly by its N terminus. The low activity of TFB2 in promoter opening and transcription can be partially relieved by transcription factor E (TFE). The results indicate that the TFB N-terminal region, containing conserved Zn ribbon and B-finger motifs, is important in promoter opening and that TFE can compensate for defects in the N terminus through enhancement of promoter opening.

Download Full-text

MOLECULAR CLONING OF THE HUMAN GENE FOR VON WILLEBRAND FACTOR AND IDENTIFICATION OF THE TRANSCRIPTION INITIATION SITE

10.1055/s-0038-1642830 ◽

1987 ◽

Author(s):

Corolyn J Collins ◽

Richard B Levene ◽

Christina P Ravera ◽

Marker J Dombalagian ◽

David M Livingston ◽

...

Keyword(s):

Endothelial Cells ◽

Transcription Start Site ◽

Von Willebrand Factor ◽

Transcription Initiation ◽

Promoter Region ◽

Start Site ◽

Transcription Start ◽

Von Willebrand's Disease ◽

Von Willebrand ◽

Willebrand Factor

Most patients with von Willebrand's disease appear to have a defect affecting the level of expression of the von Willebrand factor (vWf) gene. Thus, an understanding of the pathogenesis of von Willebrand's disease will require an analysis of the structure and function of the vWf gene in normals and in patients. To begin such analyses, we have screened a human genomic cosmid library with probes obtained from vWf cDNA and isolated a colinear segment spanning ≈175 kb in five overlapping clones. This segment extends ≈25 kb upstream and ≈5 kb downstream of the transcription start and stop sites for vWf mRNA, implying the vWf gene has a length of ≈150 kb. Within one of these clones, the vWf transcription initiation sites have been mapped. A portion of the promoter region has been sequenced, revealing a typical TATA box, a downstream CCAAT box, and a perfect downstream repeat of the 8 base pairs containing the major transcription start site. Primer extension analysis suggests that sequences contained within the downstream repeat of the transcription start site may be used as minor initiation sites in endothelial cells. Transfection studies are underway to evaluate the role of sequences within this promoter region in gene regulatory activity. Comparative restriction analyses of cloned and chromosomal DNA segments strongly suggests that no major alterations ocurred during cloning and that there is only one complete copy of the vWf gene in the human haploid genome. Similar analyses of DNA from vWf-expressing endothelial cells and non-expressing white blood cells suggests that no major rearrangements are associated with vWf gene expression. Finally, cross hybridization patterns among seven mammalian species suggests a strong conservation of genomic sequences encoding the plasma portion of vWf, but a lower degree of conservation of sequences encoding the N terminal region of provWf.

Download Full-text

Transcription by Methanothermobacter thermautotrophicus RNA Polymerase In Vitro Releases Archaeal Transcription Factor B but Not TATA-Box Binding Protein from the Template DNA

Journal of Bacteriology ◽

10.1128/jb.186.18.6306-6310.2004 ◽

2004 ◽

Vol 186 (18) ◽

pp. 6306-6310 ◽

Cited By ~ 8

Author(s):

Yunwei Xie ◽

John N. Reeve

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Transcription Initiation ◽

Tata Box ◽

Factor B ◽

Methanothermobacter Thermautotrophicus ◽

Archaeal Transcription ◽

Tata Box Binding Protein ◽

Template Dna

ABSTRACT Transcription initiation in Archaea requires the assembly of a preinitiation complex containing the TATA- box binding protein (TBP), transcription factor B (TFB), and RNA polymerase (RNAP). The results reported establish the fate of Methanothermobacter thermautotrophicus TBP and TFB following transcription initiation by M. thermautotrophicus RNAP in vitro. TFB is released after initiation, during extension of the transcript from 4 to 24 nucleotides, but TBP remains bound to the template DNA. Regulation of archaeal transcription initiation by a repressor competition with TBP for TATA-box region binding must accommodate this observation.

Download Full-text