Two Site Contact of Elongating Transcripts to Phage T7 RNA Polymerase at C-terminal Regions

Shen, Haihong; Kang, Changwon

doi:10.1074/jbc.m008616200

Cited by 12 publications

(9 citation statements)

References 24 publications

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“…Elongation complexes of biotinylated templates were obtained by stepwise walking, as described (12). The initial 80-l reaction mixture containing 40 mM Tris⅐HCl, pH 7.9, 6 mM MgCl 2 , 100 mM KCl, 10 mM DTT, 10 pmol biotinylated DNA template, 10 pmol T7 RNA polymerase (Amersham Pharmacia Biosciences), 50 M ATP, 50 M GTP, and 5 M CTP was incubated at room temperature for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Sequential multiple functions of the conserved sequence in sequence-specific termination by T7 RNA polymerase

Sohn

Kang²

2004

Proc. Natl. Acad. Sci. U.S.A.

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Escherichia coli rrnB terminator t1 contains an RNA hairpin-dependent (class I) and a sequence-specific (class II) termination signal. The latter consists of an 8-bp conserved sequence (CS), TATCTGTT, immediately followed by an 8-bp T rich sequence. In this study, elongation complexes of T7 RNA polymerase at various positions of the class II signal and several mutant signals were obtained by stepwise walking on immobilized DNA templates free of the class I signal. Multiple CS-associated conformational changes were observed, starting at the beginning of the signal and occurring sequentially. When the complexes reach the first base pair of the CS-DNA duplex, which is downstream of the RNA-DNA heteroduplex, their stability, as measured by time-course retention of radiolabeled transcripts, markedly decreases. Further elongation leads to an abrupt change in polymerase-RNA interaction. Crosslinking of the polymerase to a 4-thio-UMP incorporated into RNA 8 nucleotides upstream of the 3 end and just upstream of the heteroduplex is initially strong but diminishes when the polymerase reaches the fourth base pair of the CS. After a further 7-nt elongation, the exposed single-stranded region of nontemplate strand is contracted; RNA in the upstream half of the heteroduplex becomes dissociated, and the CS-DNA duplex is reformed. During the next 5-nt elongation before termination, the CS duplex is prevented from translocation, and the contracted transcription bubble expands only downstream. These findings suggest that the CS duplex plays essential roles by successively binding to polymerase both downstream and upstream of the heteroduplex.photocross-linking ͉ class II termination ͉ stepwise walking of RNA polymerase ͉ RNA-protein interaction ͉ elongation complex stability B acterial RNA polymerases terminate transcription by forming an RNA hairpin followed by a U rich sequence, encoded by a class I termination signal. In contrast, bacteriophage T7 and SP6 RNA polymerases terminate at both class I signals and class II termination signals, the latter consisting of a conserved sequence (CS), HATCTGTT (H designating A, C, or T), followed by a T rich sequence. The class II termination signals found in the Escherichia coli rrnB operon (1), the human preproparathyroid hormone gene (2), and vesicular stomatitis virus DNA (3) terminate phage RNA polymerase transcription, but a CS alone, without a T rich sequence, in the concatamer junction of replicating T7 DNA only causes RNA polymerase to pause (4).The class II sequence-specific termination differs from the class I hairpin-forming termination in several aspects. (i) Class II termination does not depend on formation of an RNA secondary structure; incorporation of IMP in place of GMP into RNA does not affect termination (1, 5). (ii) Class II termination is highly specific in sequence, requiring a perfect CS duplex; introduction of mutations and mismatches abolishes termination (1,5,6). (iii) Introduction of a nick into T7 or SP6 RNA polymerase resulting in N-terminal 20-kDa and C-termi...

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Section: Methodsmentioning

confidence: 99%

Sequential multiple functions of the conserved sequence in sequence-specific termination by T7 RNA polymerase

Sohn

Kang²

2004

Proc. Natl. Acad. Sci. U.S.A.

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“…The proposed trajectory results in few clashes, is consistent with much of the biochemical and genetic data, and is not consistent with the RNA-exit pathway suggested by the structure of the T7 RNAP initiation complex (12). In an independent study, Shen and Kang (19) mapped T7 RNAP-RNA crosslinks from either the derivatized 3Ј end of the nascent RNA or from the Ϫ9 position of the nascent RNA in several stalled elongation complexes. Their results are in agreement with those of Temiakov et al (15).…”

mentioning

confidence: 57%

T7 RNA polymerase transcription complex: What you see is not what you get

Severinov

2000

Proc. Natl. Acad. Sci. U.S.A.

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“…We assumed that 9 residues at the 3Ј-end (from positions T-8 to T) cannot participate in the formation of a secondary structure because 7 or 8 residues at the 3Ј-end are engaged in an RNA-DNA hybrid (21,22), and the 9th residue from the 3Ј-end is in contact with T7 RNA polymerase (23,24). A stable terminator hairpin structure with an 11-bp stem (⌬G ϭ Ϫ14.8 kcal/ mol) was estimated for the T pause complex (Fig.…”

Section: Stalling or Slowing Down At Position T-3 Facilitatesmentioning

confidence: 99%

“…A, a putative terminator RNA secondary structure representing the most stable one for the T pause complex predicted by the mfold program. Nine nucleotides at the 3Ј-end were excluded from formation of secondary structures because 7 or 8 nucleotides (dashed box) are secluded in the RNA-DNA hybrid (21,22), and the 9th residue (solid box) from the 3Ј-end makes contact with T7 RNA polymerase (23,24). The cleavage sites of RNases A (dashed arrows), T1 (solid arrows), and V1 (arrowheads) are shown with arrows or arrowheads, the length of which is roughly proportional to the cleavage efficiency.…”

Section: Rna Structure Responsible For Cis-acting Antitermination-mentioning

confidence: 99%

Opposite Consequences of Two Transcription Pauses Caused by an Intrinsic Terminator Oligo(U)

Lee

Kang

2011

Journal of Biological Chemistry

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The RNA oligo(U) sequence, along with an immediately preceding RNA hairpin structure, is an essential cis-acting element for bacterial class I intrinsic termination. This sequence not only causes a pause in transcription during the beginning of the termination process but also facilitates transcript release at the end of the process. In this study, the oligo(U) sequence of the bacteriophage T7 intrinsic terminator T, rather than the hairpin structure, induced pauses of phage T7 RNA polymerase not only at the termination site, triggering a termination process, but also 3 bp upstream, exerting an antitermination effect. The upstream pause presumably allowed RNA to form a thermodynamically more stable secondary structure rather than a terminator hairpin and to persist because the 5-half of the terminator hairpin-forming sequence could be sequestered by a farther upstream sequence via sequence-specific hybridization, prohibiting formation of the terminator hairpin and termination. The putative antiterminator RNA structure lacked several base pairs essential for termination when probed using RNases A, T1, and V1. When the antiterminator was destabilized by incorporation of IMP into nascent RNA at G residue positions, antitermination was abolished. Furthermore, antitermination strength increased with more stable antiterminator secondary structures and longer pauses. Thus, the oligo(U)-mediated pause prior to the termination site can exert a cis-acting antitermination activity on intrinsic terminator T, and the termination efficiency depends primarily on the termination-interfering pause that precedes the termination-facilitating pause at the termination site.Multisubunit bacterial RNA polymerase transcription terminates upon various termination signals, causing disassembly of elongation complexes (ECs) 2 with or without the assistance of termination factors (1, 2). The most common class of intrinsic (factor-independent) termination signal relies on an oligo(U) sequence in the RNA preceded by an RNA hairpin structure (class I intrinsic termination signal) (3). The termination mechanism of class I signals is shared by virtually all multisubunit bacterial and single-subunit phage RNA polymerases. However, it is distinct from the mechanism of class II intrinsic termination, which is caused by specific DNA sequence recognition of single-subunit phage T7 RNA polymerase (4).The oligo(U) sequence allows ECs to pause prior to the release of RNA transcripts (5, 6). This transcription pause has been shown to be the first signal for the class I termination pathway, providing additional time for the formation of a terminator RNA hairpin that opens or unwinds the RNA-DNA hybrid at an upstream region, shortening and weakening the hybrid (5,7,8). Because the remaining downstream part of the hybridized region consists mostly of rU:dA base pairs, it becomes thermodynamically unstable, facilitating the release of nascent RNA transcripts (9).In this study, an opposite effect of the oligo(U)-mediated pause was found with a typical class I...

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Two Site Contact of Elongating Transcripts to Phage T7 RNA Polymerase at C-terminal Regions

Cited by 12 publications

References 24 publications

Sequential multiple functions of the conserved sequence in sequence-specific termination by T7 RNA polymerase

Sequential multiple functions of the conserved sequence in sequence-specific termination by T7 RNA polymerase

T7 RNA polymerase transcription complex: What you see is not what you get

Opposite Consequences of Two Transcription Pauses Caused by an Intrinsic Terminator Oligo(U)

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