Marking the start site of RNA polymerase III transcription: the role of constraint, compaction and continuity of the transcribed DNA strand

Grove, Anne; Adessa, Morgan S.; Geiduschek, E. Peter; Kassavetis, George A.

doi:10.1093/emboj/21.4.704

Cited by 16 publications

(39 citation statements)

References 53 publications

(90 reference statements)

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“…This possibility clearly applies to the previously analyzed TATA-less SUP4 tRNA gene, for which alternative placements of TFIIIB by TFIIIC lead to heterogeneity in selection of the transcriptional start site (39,40). However, this does not appear to be the case for the TATA-containing SNR6 (U6) gene; initiation occurs exclusively at bp ϩ1 on U6 DNA templates with an intact transcribed strand (25).…”

Section: Implications Of the Complexity Of Protein Cross-linking At 30mentioning

confidence: 91%

“…DNA Templates and Probes-The 198-bp (Ϫ60 to ϩ138) pU6 R boxBderived transcription template has been described (25). Fully duplex and heteroduplex bubble-containing 86-bp (Ϫ56 to ϩ30) photochemical cross-linking probes all differ from pU6 R boxB at three positions: an upstream (Ϫ29) A 3 G substitution to generate a TGTA mutant TATA box for TFIIIB binding in a single orientation with the TBP variant, TBPm3 (26) and two downstream (ϩ8 and ϩ9) A 3 T substitutions (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…1A). These substitutions were generated by PCR with mutagenic primers using the above-specified 198-bp transcription template (25) and variants of this template that were previously used for making transcription templates with short heteroduplex bubbles (27). Bottom (transcribed) strands that served as templates for primer-directed incorporation of the photoreactive deoxyribonucleotide 5-(NЈ-[p-azidobenzoyl]-3-aminoallyl)-dUTP (28) with adjacent 32 P-labeled deoxyribonucleotides (essentially as described in Ref.…”

Section: Methodsmentioning

confidence: 99%

“…Bottom (transcribed) strands that served as templates for primer-directed incorporation of the photoreactive deoxyribonucleotide 5-(NЈ-[p-azidobenzoyl]-3-aminoallyl)-dUTP (28) with adjacent 32 P-labeled deoxyribonucleotides (essentially as described in Ref. 29) and heteroduplex formation were generated by PCR in which the primer for the bottom strand contained a ribonucleotide at its 3Ј end for subsequent alkaline hydrolysis and denaturing gel isolation (25). One half of each duplex photoreactive probe was denatured at 94°C in the presence of a 3-fold molar excess of bottom strands altered in sequence to form either a Ϫ9/Ϫ5 or a ϩ2/ϩ6 heteroduplex bubble (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…One anticipates that the pattern of cross-linking at bp ϩ2/ϩ3 would change when the promoter opens because a fully opened promoter complex pulls an additional 4 -5 bp of downstream DNA into polymerase (25), and open complex formation should ac-cordingly shift the location of bp ϩ2 and ϩ3 relative to protein.…”

Section: Implications Of the Complexity Of Protein Cross-linking At 30mentioning

confidence: 99%

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The Role of Transcription Initiation Factor IIIB Subunits in Promoter Opening Probed by Photochemical Cross-linking

Kassavetis

Han

Naji

et al. 2003

Journal of Biological Chemistry

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The core transcription initiation factor (TF) IIIB recruits its conjugate RNA polymerase (pol) III to the promoter and also plays an essential role in promoter opening. TFIIIB assembled with certain deletion mutants of its Brf1 and Bdp1 subunits is competent in pol III recruitment, but the resulting preinitiation complex does not open the promoter. Whether Brf1 and Bdp1 participate in opening the promoter by direct DNA interaction (as subunits of bacterial RNA polymerases do) or indirectly by their action on pol III has been approached by site-specific photochemical protein-DNA cross-linking of TFIIIB-pol III-U6 RNA gene promoter complexes. Brf1, Bdp1, and several pol III subunits can be crosslinked to the nontranscribed strand of the U6 promoter at base pair ؊9/؊8 and ؉2/؉3 (relative to the transcriptional start as ؉1), respectively the upstream and downstream ends of the DNA segment that opens up into the transcription bubble. Cross-linking of Bdp1 and Brf1 is detected at 0°C in closed preinitiation complexes and at 30°C in complexes that are partly open, but also it is detected in mutant TFIIIB-pol III-DNA complexes that are unable to open the promoter. In contrast, promoter opening-defective TFIIIB mutants generate significant changes of cross-linking of polymerase subunits. The weight of this evidence argues in favor of an indirect mode of action of TFIIIB in promoter opening.Bacterial RNA polymerase holoenzymes locate their cognate promoters through subunits that recognize specific DNA motifs located upstream of transcriptional start sites. subunits also participate in subsequent steps of the reaction sequence that generates an RNA chain-elongating transcription complex. For example: 1) 54 is directly involved in initiating promoter opening next to its Ϫ12 DNA-binding site (1); 2) 70 participates directly in initiating promoter opening and binds sequence-specifically to the nontranscribed strand of the upstream segment of the transcription bubble in the open promoter complex (2-5); and 3) 70 also participates in promoterproximal pausing events that are required for assembly of promoter-specific termination-resistant elongation complexes (6 -8). In addition, 70 and 54 also mediate effects of proteins that activate transcription by allowing or accelerating promoter opening (9, 10).Eukaryotic nuclear RNA polymerases locate their promoters through DNA-bound complexes of core transcription initiation factors. Yeast RNA polymerase (pol) 1 III is brought to its promoters primarily by interacting with its transcription factor TFIIIB, which is composed of subunits TBP, Brf1, and Bdp1 (previously called BЉ or TFIIIB90). A homologous assembly operates at most human pol III promoters, whereas a paralogous assembly containing Brf2 (previously called BRFU or TFIIIB50) in place of Brf1 functions at a specialized subgroup of promoters that require participation of the SNAP C DNAbinding complex and its activators (reviewed in Refs. 11-13; see Ref. 14 for a new TFIIIB subunit nomenclature).TFIIIB also participates in two...

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Section: Implications Of the Complexity Of Protein Cross-linking At 30mentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Implications Of the Complexity Of Protein Cross-linking At 30mentioning

confidence: 99%

See 3 more Smart Citations

The Role of Transcription Initiation Factor IIIB Subunits in Promoter Opening Probed by Photochemical Cross-linking

Kassavetis

Han

Naji

et al. 2003

Journal of Biological Chemistry

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Long-term RNA interference from optimized siRNA expression constructs in adult mice

Wooddell

Hout

Reppen

et al. 2005

Biochemical and Biophysical Research Communications

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The Transcription Bubble of the RNA Polymerase–Promoter Open Complex Exhibits Conformational Heterogeneity and Millisecond-Scale Dynamics: Implications for Transcription Start-Site Selection

Robb

Cordes

Hwang

et al. 2013

Journal of Molecular Biology

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Bacterial transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 bp around the transcription start site and forms a single-stranded “transcription bubble” within a catalytically active RNAP–DNA open complex (RPo). There is significant flexibility in the transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5′ end of RNA transcripts and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RPo. The flexibility may occur in the positioning of the transcription bubble prior to RNA synthesis and may reflect bubble expansion (“scrunching”) or bubble contraction (“unscrunching”). Here, we assess the presence of dynamic flexibility in RPo with single-molecule FRET (Förster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RPo using different FRET rulers and labeling positions. An analysis of FRET distributions of RPo using burst variance analysis reveals conformational fluctuations in RPo in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the transcription start sites, in a way that can be described by repositioning of the single-stranded transcription bubble relative to the RNAP active site within RPo. Our study marks the first experimental observation of conformational dynamics in the transcription bubble of RPo and indicates that DNA dynamics within the bubble affect the search for transcription start sites.

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Marking the start site of RNA polymerase III transcription: the role of constraint, compaction and continuity of the transcribed DNA strand

Cited by 16 publications

References 53 publications

The Role of Transcription Initiation Factor IIIB Subunits in Promoter Opening Probed by Photochemical Cross-linking

The Role of Transcription Initiation Factor IIIB Subunits in Promoter Opening Probed by Photochemical Cross-linking

Long-term RNA interference from optimized siRNA expression constructs in adult mice

The Transcription Bubble of the RNA Polymerase–Promoter Open Complex Exhibits Conformational Heterogeneity and Millisecond-Scale Dynamics: Implications for Transcription Start-Site Selection

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