Role of the Sulfolobus shibatae viral T6 initiator in conferring promoter strength and in influencing transcription start site selection

Archaeal promoters contain a TATA-box, an adjacent upstream TFB-recognition element (BRE), and a downstream initiator (INR) region from which transcription originates. While the contribution of A-box and BRE to promoter strength is well established, the role of DNA sequences within the INR region and its vicinity on transcription efficiency and start site selection remains unclear. Here, I demonstrate using the strong Sulfolobus shibatae viral T6 promoter that either substitution of its natural sequence from –17 and beyond with plasmid DNA or introduction of point transversion mutations at +3, –2, –4, and –5 positions reduce promoter strength dramatically, whereas +1, –1, and –2 mutations influence the transcription start site. These data therefore reveal that the INR region plays a role as important as the BRE and the A-box in T6 gene transcription. Key words: Archaea, transcription, initiator (INR), Sulfolobus shibatae, core promoter.

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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.

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Analysis of the role of human TFIIB in transcription start site selection

Biochemical Society Transactions ◽

10.1042/bst027a093c ◽

1999 ◽

Vol 27 (3) ◽

pp. A93-A93

Author(s):

Nicola A. Hawkes ◽

Stefan G. E. Roberts

Keyword(s):

Transcription Start Site ◽

Site Selection ◽

Start Site ◽

Transcription Start

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The Role of XPB/Ssl2 dsDNA Translocase Processivity in Transcription Start-site Scanning

Journal of Molecular Biology ◽

10.1016/j.jmb.2021.166813 ◽

2021 ◽

pp. 166813

Author(s):

Eric J. Tomko ◽

Olivia Luyties ◽

Jenna K. Rimel ◽

Chi-Lin Tsai ◽

Jill O. Fuss ◽

...

Keyword(s):

Transcription Start Site ◽

Start Site ◽

Transcription Start

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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).

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Influence of cis Element Arrangement on Promoter Strength in Trichoderma reesei

Applied and Environmental Microbiology ◽

10.1128/aem.01742-17 ◽

2017 ◽

Vol 84 (1) ◽

Cited By ~ 12

Author(s):

Daniel P. Kiesenhofer ◽

Robert L. Mach ◽

Astrid R. Mach-Aigner

Keyword(s):

Trichoderma Reesei ◽

Transcription Start Site ◽

Binding Sites ◽

Inverted Repeat ◽

Promoter Activity ◽

Promoter Strength ◽

Start Site ◽

Transcription Start ◽

Content Type ◽

Sheer Number

ABSTRACTTrichoderma reeseican produce up to 100 g/liter of extracellular proteins. The major and industrially relevant products are cellobiohydrolase I (CBHI) and the hemicellulase XYNI. The genes encoding both enzymes are transcriptionally activated by the regulatory protein Xyr1. The first 850 nucleotides of thecbh1promoter contain 14 Xyr1-binding sites (XBS), and 8 XBS are present in thexyn1promoter. Some of these XBS are arranged in tandem and others as inverted repeats. One suchciselement, an inverted repeat, plays a crucial role in the inducibility of thexyn1promoter. We investigated the impact of the properties of suchciselements by shuffling them by insertion, exchange, deletion, and rearrangement ofciselements in both thecbh1andxyn1promoter. A promoter-reporter assay using theAspergillus nigergoxAgene allowed us to measure changes in the promoter strength and inducibility. Most strikingly, we found that an inverted repeat of XBS causes an important increase incbh1promoter strength and allows induction by xylan or wheat straw. Furthermore, evidence is provided that the distances ofciselements to the transcription start site have important influence on promoter activity. Our results suggest that the arrangement and distances ofciselements have large impacts on the strength of thecbh1promoter, whereas the sheer number of XBS has only secondary importance. Ultimately, the biotechnologically importantcbh1promoter can be improved byciselement rearrangement.IMPORTANCEIn the present study, we demonstrate that the arrangement ofciselements has a major impact on promoter strength and inducibility. We discovered an influence on promoter activity by the distances ofciselements to the transcription start site. Furthermore, we found that the configuration ofciselements has a greater effect on promoter strength than does the sheer number of transactivator binding sites present in the promoter. Altogether, the arrangement ofciselements is an important factor that should not be overlooked when enhancement of gene expression is desired.

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