Two modes of transcription initiation in vitro at the rrnB P1 promoter of Escherichia coli.

Borukhov, Sergei; Sagitov, V; Josaitis, Cathleen A.; Gourse, Richard L.; Goldfarb, Alex

doi:10.1016/s0021-9258(19)49487-8

Cited by 64 publications

(5 citation statements)

References 27 publications

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“…On the other hand, during abortive cycling with GTP and ATP, the active site of the enzyme can move up to +7 from the start site (+1 arrow in Figure 1) and the short transcripts are thought to be released from T7 RNAP ternary complexes without enzyme dissociation from the template. Ternary complexes in the slippage mode may be more stable than those in the abortive mode (Borukhov et al, 1992;Sousa et al, 1992).…”

Section: Resultsmentioning

confidence: 97%

Probing the Mechanisms of T7 RNA Polymerase Transcription Initiation Using Photochemical Conjugation of Psoralen to a Promoter

Sastry

Ross

1997

Biochemistry

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We have dissected the steps in T7 RNA polymerase transcription initiation using psoralen cross-linking. DNA templates containing cross-links at either -14/-13, -2/-1, or -4/-3 were constructed. These cross-links are within the DNA-contacting region in the initiation complex. A cross-link at -2/-1 did not affect T7 RNA polymerase binding affinity, whereas a cross-link at -14/-13 reduced binding affinity by less than 2-fold. Transcription initiation was completely blocked by cross-links at -14/-13 or at -2/-1. A cross-link at -4/-3 inhibited neither binding nor the first RNA phosphodiester bond but greatly inhibited further RNA chain extension. Circular dichroism spectroscopy revealed that DNA melting in the -4/-3 cross-link was greatly inhibited, indicating that inhibition of RNA chain extension was a melting defect. Transcription shutoff on the -14/-13 cross-link may be due to inhibition of conformational changes in the polymerase-DNA complex. Because the -2/-1 cross-link is immediately upstream of the start site (+1), open complex formation may have been completely inhibited by this cross-link, accounting for the shutoff of transcription. Thus, depending on their location, psoralen cross-links affected different steps in the initiation process. We propose that promoter melting is progressive and that melting of one or two bp upstream of the +1 site is sufficient for formation of the first phosphodiester bond while further RNA chain extension within the promoter depends on greater upstream melting of the promoter, which may be required for stabilization of the initiation complex.

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Section: Resultsmentioning

confidence: 97%

Probing the Mechanisms of T7 RNA Polymerase Transcription Initiation Using Photochemical Conjugation of Psoralen to a Promoter

Sastry

Ross

1997

Biochemistry

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“…This caveat is pertinent because E. coli RNA polymerase can pause in the region of positions 15-25 to yield transcripts that are considered paused products, despite the fact that the σ subunit has not been released from these promoters (30). Additionally, the phenomenon known as "primer shifting" can lead to longer abortive products that are no longer precisely complementary to the initial transcribed region of the template sequence (43)(44)(45). These findings prompted us, especially in the case of T5 N25 antiDSR where abortive products extend to 15 nt, to obtain further verification as to whether the short RNAs were true abortive transcripts.…”

Section: Resultsmentioning

confidence: 99%

“…This is an unprecedented reaction with DNA-dependent RNA polymerases. It does not resemble primer shifting, which requires a template region upstream of position 1 that is complementary to the initiating primer ( , ). It also differs from the “slipping” reaction demonstrated by others in two respects: (1) it does not form a long sequence of C residues (), and (2) it slips over an internal CC dinucleotide repeat rather than the three identical template residues normally required ().…”

Section: Discussionmentioning

confidence: 99%

In Vitro Studies of Transcript Initiation by Escherichia coli RNA Polymerase. 1. RNA Chain Initiation, Abortive Initiation, and Promoter Escape at Three Bacteriophage Promoters

Hsu

Kane

et al. 2003

Biochemistry

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RNA chain initiation and promoter escape is the latter stage of transcription initiation. This stage is characterized by several well-defined biochemical events: synthesis and release of short RNA products ranging 2 to 15 nucleotides in length, release of the sigma subunit from the enzyme-promoter complex, and initial translocation of the polymerase away from the promoter. In this paper, we report the use of a steady-state transcription assay with [gamma-(32)P]ATP labeling to subject the RNA chain initiation-promoter escape reaction to quantitative analysis. The specific parameters we follow to describe the chain initiation-promoter escape process include the abortive and productive rates, the abortive probability, the abortive:productive ratio, and the maximal size of the abortive product. In this study, we measure these parameters for three bacteriophage promoters transcribed by Escherichia coli RNA polymerase: T7 A1, T5 N25, and T5 N25(antiDSR). Our studies show that all three promoters form substantial amounts of abortive products under all conditions we tested. However, each of the promoters shows distinct differences from the others when the various parameters are compared. At 100 microM NTP, in a 10 min reaction, the abortive and productive yields are 87 and 13%, respectively, for T7 A1; 97 and 3%, respectively, for T5 N25; and 99.4 and 0.6%, respectively, for T5 N25(antiDSR). These values correspond to approximately 7, 32, and 165 abortive transcripts per productive transcript for the three promoters, respectively. The yield of most of the abortive products is not affected by the elevated concentration of the NTP substrate corresponding to the next template-specified nucleotide; hence, abortive products are not normally formed through a simple process of "kinetic competition". Instead, formation of abortive products appears to be determined by intrinsic DNA signals embedded in the promoter recognition region and the initial transcribed sequence region of each promoter.

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“…The effect of mutations in the interdomain linker of the R subunit on UP element-dependent rrnB P1 transcription was tested in vitro. Closed circular plasmids were used as templates because the rrnB P1 promoter on linear DNA can form a stable open complex only in the presence of initiating nucleotides ATP and CTP (29), and the complex formed in the presence of ATP and CTP may not be a natural intermediate of rrnB P1 transcription initiation (30). As can be seen in Figure 4, the overall responses of the UP element-dependent rrnB P1 transcription to various linker mutations were very similar to those of the CRP-dependent lac P1 transcription (Figure 3), including the severe defect in activated transcription for ∆6, ∆9, and ∆12 deletions, the higher transcription activity of the Ω3 derivative compared with other insertion derivatives (Ω1, Ω2, and Ω4), and the substantial and complete loss of activated transcription in the Gly and amphi derivatives, respectively.…”

Section: Effect Of Linkermentioning

confidence: 99%

Structural Requirements for the Interdomain Linker of α Subunit of Escherichia coli RNA Polymerase

2000

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The carboxy-terminal domain of the alpha subunit of Escherichia coli RNA polymerase, which is connected with the core part of RNA polymerase through a long flexible linker, plays decisive roles in transcription activation by directly interacting with a large number of transcription factors and upstream (UP) element DNA. Here we constructed a set of mutant RNA polymerases, each containing a mutant alpha subunit with an altered interdomain linker. Deletion of three amino acids from the linker exhibited 50% inhibition of cAMP receptor protein- (CRP-) dependent lac P1 transcription. Deletion of six amino acids completely knocked out the activity. Insertion of three amino acids did not affect the activity, whereas 40-60% inhibition was observed after insertion of one, two, or four amino acids. Substitution of 10 consecutive glycine residues resulted in nearly 90% reduction of the CRP-dependent activity, whereas 50% activity was retained after substitution of 10 proline residues or a sequence expected to form a strong alpha-helix. Essentially the same results were obtained with UP element-dependent rrnB P1 transcription. These observations altogether suggest that (i) sufficient length of the interdomain linker is required for transcription activation mediated by the alpha carboxy-terminal domain, (ii) the linker is not totally unstructured but has structural and torsional preferences to facilitate positioning of the carboxy-terminal domain to a proper location for the interaction with CRP and UP element, and (iii) CRP-dependent activation and UP element-dependent activation share a common intermediary state in which the positioning of the alpha carboxy-terminal domain is of primary importance.

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Two modes of transcription initiation in vitro at the rrnB P1 promoter of Escherichia coli.

Cited by 64 publications

References 27 publications

Probing the Mechanisms of T7 RNA Polymerase Transcription Initiation Using Photochemical Conjugation of Psoralen to a Promoter

Probing the Mechanisms of T7 RNA Polymerase Transcription Initiation Using Photochemical Conjugation of Psoralen to a Promoter

In Vitro Studies of Transcript Initiation by Escherichia coli RNA Polymerase. 1. RNA Chain Initiation, Abortive Initiation, and Promoter Escape at Three Bacteriophage Promoters

Structural Requirements for the Interdomain Linker of α Subunit of Escherichia coli RNA Polymerase

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