Abortive initiation and long RNA synthesis

Munson, Lianna M.; Reznikoff, William S.

doi:10.1021/bi00511a003

Cited by 96 publications

(71 citation statements)

References 14 publications

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“…These complexes retain cr and usually are unstable; they frequently release the nascent RNA, revert to the binary open complex, and reinitiate another RNA molecule. This repeating cycle of RNA initiation, abortive release, and reinitiation has been observed at all promoters examined by Levin et al (1987) at low substrate concentrations; for some promoters, abortive cycling is quite pronounced even at optimal substrate concentrations (Carpousis and Gralla 1980;Munson and Reznikoff 1981;Liao et al 1987). Eventually, the initiating complex escapes from this abortive cycle and undergoes a major transition when Figure 1.…”

Section: Icoztesponding Authormentioning

confidence: 76%

Pseudo-templated transcription by Escherichia coli RNA polymerase at a mutant promoter.

Jacques¹,

Susskind²

1990

Genes Dev.

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A G--~ T mutation at the start-point of transcription of the phage P22 sat promoter (sor+ 1T) causes a novel defect in promoter clearance by Escherichia coli RNA polymerase (RNAP) in vitro. Under standard transcription conditions, in the presence of high concentrations of all four NTPs, the predominant products from this promoter are poly(U) chains of varying length. Because the mutation creates a run of four T : A base-pairs from -1 to + 3 (TGTT --, TTTT), we propose that synthesis of poly(U) is pseudo-templated by the A4 stretch on the template strand. G --* A and G --* C mutations at position + 1 do not cause pseudo-templated transcription. Several molecules of poly(U) are produced and released per sar+ IT promoter-polymerase complex without dissociation of RNAP from the template DNA. The exponential relationship between yield and size of individual poly(U) species indicates that there is a constant probability that another U residue will be added to the nascent chain. Presumably, pseudo-templated transcription occurs by a slippage (stuttering) mechanism like that proposed to explain certain kinds of RNA editing in eukaryotic viral mRNAs.[Key Words: E. coli RNA polymerase; pseudo-templated transcription; Sar + 1 T]Received May 11, 1990; revised version accepted July 31, 1990.Initiation of transcription by E. coli RNA polymerase (RNAP) is a complex process that can be divided into several steps: (1) initial binding of the polymerase holoenzyme (core enzyme + cr) to form an unstable "closed" complex; (2) isomerization to form a stable "open" complex in which the DNA around the start point of transcription is unwound; (3) initiation of RNA synthesis; and (4) promoter clearance, which includes release of the promoter and (r subunit, and formation of a stable ternary complex of DNA, nascent RNA, and core enzyme.Much attention has focused on the early steps leading to formation of the open complex because the rate of open complex formation is clearly a primary determinant of promoter strength and is often the target of control by repressors and activators (for review, see Hoopes andYager and von Hippel 1987). Footprinting studies of open complexes on several different promoters show that the polymerase is in close proximity to the promoter DNA between about -50 and +20 and that the region from -9 to +3 is unwound. Within the protected region are the two stretches of DNA sequence that are most highly conserved among promoters, the -35 and -10 regions. The importance of these sequences is underscored by analyses of promoter-down and promoter-up mutations, most of which cluster in the conserved hexamers 5'-ICoztesponding author.

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Section: Icoztesponding Authormentioning

confidence: 76%

Pseudo-templated transcription by Escherichia coli RNA polymerase at a mutant promoter.

Jacques¹,

Susskind²

1990

Genes Dev.

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“…Differences at the 5' ends of transcripts can affect transcript stability (4,16) or control the formation of secondary structures that inhibit translational initiation as seen with pyrC (2,3). Switching also can result in the selection of start sites with different potentials for abortive initiation (14) and transcriptional slippage during initiation (8). Both of these processes can greatly influence the frequency of productive initiation at a particular promoter (9,26).…”

Section: Methodsmentioning

confidence: 99%

Effects of transcriptional start site sequence and position on nucleotide-sensitive selection of alternative start sites at the pyrC promoter in Escherichia coli

Liu

Turnbough

1994

J Bacteriol

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In Escherichia coli, expression of the pyrC gene is regulated primarily by a translational control mechanism based on nucleotide-sensitive selection of transcriptional start sites at the pyrC promoter. When intracellular levels of CTP are high, pyrC transcripts are initiated predominantly with CTP at a site 7 bases downstream of the Pribnow box. These transcripts form a stable hairpin at their 5' ends that blocks ribosome binding. When the CTP level is low and the GTP level is high, conditions found in pyrimidine-limited cells, transcripts are initiated primarily with GTP at a site 9 bases downstream of the Pribnow box. These shorter transcripts are unable to form a hairpin at their 5' ends and are readily translated. In this study, we examined the effects of nucleotide sequence and position on the selection of transcriptional start sites at the pyrC promoter. We characterized promoter mutations that systematically alter the sequence at position 7 or 9 downstream of the Pribnow box or vary the spacing between the Pribnow box and wild-type transcriptional initiation region. The results reveal preferences for particular initiating nucleotides (ATP 2 GTP > UTP > > CTP) and for starting positions downstream of the Pribnow box (7 >> 6 and 8 > 9 > 10). The results indicate that optimal nucleotide-sensitive start site switching at the wild-typepyrC promoter is the result of competition between the preferred start site (position 7) that uses the poorest initiating nucleotide (CTP) and a weak start site (position 9) that uses a good initiating nucleotide (GTP). The sequence of the pyrC promoter also minimizes the synthesis of untranslatable transcripts and provides for maximum stability of the regulatory transcript hairpin. In addition, the results show that the effects of the mutations on pyrC expression and regulation are consistent with the current model for translational control. Possible effects of preferences for initiating nucleotides and start sites on the expression and regulation of other genes are discussed.In Eschenchia coli and Salmonella typhimurium, six unlinked genes and operons designated carAB, pyrBI, pyrC, pyrD, pyrE, and pyrF encode the six enzymes that catalyze the de novo synthesis of UMP, the precursor of all pyrimidine nucleotides. These genes and operons are subject to negative and noncoordinate regulation mediated by pyrimidine availability (15). Expression of the pyrC gene, which encodes the enzyme dihydroorotase, is regulated primarily by a translational control mechanism that is based on nucleotide-sensitive selection of transcriptional start sites (22,23). Transcriptional initiation at the pyrC promoter can occur at four adjacent sites (specifying UCCG) located 6 to 9 bases downstream of the Pribnow box (24). The transcripts initiated at these sites are designated U-6, C-7, C-8, and G-9. According to the model for regulation, when the intracellular level of CTP is high, C-7 transcripts are synthesized predominantly. These transcripts are poorly translated, however, because they form a stable...

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“…The problem has been posed by two seemingly contradictory observations: First, RNA products up to ~8-10 nt in length are synthesized in abortive initiation (4)(5)(6); thus the RNAP active center translocates relative to DNA in abortive initiation. Second, DNA-footprinting results indicate that there upstream boundary of the DNA segment protected by RNAP is the same in RP o and in RP itc engaged in abortive synthesis (7)(8)(9)(10); thus RNAP appears not to translocate relative to DNA in abortive initiation.…”

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confidence: 99%

Abortive Initiation and Productive Initiation by RNA Polymerase Involve DNA Scrunching

Revyakin

Liu

Ebright

et al. 2006

Science

355

447

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Using single-molecule DNA nanomanipulation, we show that abortive initiation involves DNA "scrunching"--in which RNA polymerase (RNAP) remains stationary and unwinds and pulls into itself downstream DNA--that scrunching requires RNA synthesis, and that scrunching depends on RNA length. We show further that promoter escape involves scrunching, and that scrunching occurs in most or all instances of promoter escape. Our results support existence of an obligatory stressed intermediate, with ~1 turn of additional DNA unwinding, in escape and are consistent with the proposal that stress in this intermediate provides the driving force to break RNAP-promoter and RNAP-initiation-factor interactions in escape.Transcription initiation involves a series of reactions (1-3). RNA polymerase (RNAP) binds to promoter DNA to yield an RNAP-promoter closed complex (RP c ). RNAP then unwinds ~1 turn of DNA surrounding the transcription start site to yield an RNAP-promoter open complex (RP o ). RNAP then enters into abortive cycles of synthesis and release of short RNA products as an RNAP-promoter initial transcribing complex (RP itc ) and, upon synthesis of an RNA product ~9-11 nt in length, escapes the promoter and enters into productive synthesis of RNA as an RNAP-DNA elongation complex (RD e ).The mechanism by which the RNAP active-center translocates in abortive initiation and promoter escape has remained problematic. The problem has been posed by two seemingly contradictory observations: First, RNA products up to ~8-10 nt in length are synthesized in abortive initiation (4-6); thus the RNAP active center translocates relative to DNA in abortive initiation. Second, DNA-footprinting results indicate that there upstream boundary of the DNA 1A;4,7,9-12; see also proposals for structurally unrelated single-subunit RNAP derivatives in 13-19).In previous work, we have developed a single-molecule-DNA-nanomanipulation approach that detects RNAP-dependent DNA unwinding with ~1 bp resolution and ~1 s temporal resolution, and we have applied this approach to detect and characterize RNAP-dependent promoter unwinding upon formation of RP o (Figs. 1B, S1,(20)(21)(22). In this work, we have applied this approach to test the scrunching model for RNAP-active-center translocation in abortive initiation and promoter escape (Fig. 1A;4,7,11,12). The scrunching model--and only the scrunching model--postulates changes in RNAP-dependent DNA unwinding during abortive initiation and promoter escape. Specifically, the scrunching model postulates that RNAP pulls into itself downstream DNA; for each base pair that RNAP pulls into itself, a base pair must be broken and must be maintained broken, and, correspondingly there must be one base pair of additional DNA unwinding.Our first set of experiments addressed abortive initiation occurring in complexes engaged in iterative abortive initiation [complexes prepared using subsets of nucleoside triphosphates (NTPs) insufficient to permit promoter escape and productive initiation]. Our primary experimental...

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Abortive initiation and long RNA synthesis

Cited by 96 publications

References 14 publications

Pseudo-templated transcription by Escherichia coli RNA polymerase at a mutant promoter.

Pseudo-templated transcription by Escherichia coli RNA polymerase at a mutant promoter.

Effects of transcriptional start site sequence and position on nucleotide-sensitive selection of alternative start sites at the pyrC promoter in Escherichia coli

Abortive Initiation and Productive Initiation by RNA Polymerase Involve DNA Scrunching

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