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EcoSal Plus

Domain 4:

Synthesis and Processing of Macromolecules

Promoter Escape by RNA Polymerase

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  • Author: Lilian M. Hsu1
  • Editor: Susan T. Lovett2
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Program in Biochemistry, Mount Holyoke College, South Hadley, MA 01075; 2: Brandeis University, Waltham, MA
  • Received 13 September 2007 Accepted 07 November 2007 Published 12 February 2008
  • Address correspondence to Lilian M. Hsu lhsu@mtholyoke.edu.
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  • Abstract:

    Promoter escape is the process that an initiated RNA polymerase (RNAP) molecule undergoes to achieve the initiation-elongation transition. Having made this transition, an RNAP molecule would be relinquished from its promoter hold to perform productive (full-length) transcription. Prior to the transition, this process is accompanied by abortive RNA formation—the amount and pattern of which is controlled by the promoter sequence information. Qualitative and quantitative analysis of abortive/productive transcription from several promoters and their sequence variants led to the understanding that a strong (RNAP-binding) promoter is more likely to be rate limited (during transcription initiation) at the escape step and produce abortive transcripts. Of the two subelements in a promoter, the PRR (the core Promoter Recognition Region) was found to set the initiation frequency and the rate-limiting step, while the ITS (the Initial Transcribed Sequence region) modulated the ratio of abortive versus productive transcription. The highly abortive behavior of RNAP could be ameliorated by the presence of Gre (transcript cleavage stimulatory) factor(s), linking the first step in abortive RNA formation by the initial transcribing complexes (ITC) to RNAP backtracking. The discovery that translocation during the initiation stage occurs via DNA scrunching provided the source of energy that converts each ITC into a highly unstable "stressed intermediate." Mapping all of the biochemical information onto an X-ray crystallographic structural model of an open complex gave rise to a plausible mechanism of transcription initiation. The chapter concludes with contemplations of the kinetics and thermodynamics of abortive initiation-promoter escape.

  • Citation: Hsu L. 2008. Promoter Escape by RNA Polymerase, EcoSal Plus 2008; doi:10.1128/ecosalplus.4.5.2.2

Key Concept Ranking

RNA Polymerase
0.6387286
Core Promoter
0.63004017
Upstream Promoter
0.61663264
0.6387286

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2008-02-12
2017-07-21

Abstract:

Promoter escape is the process that an initiated RNA polymerase (RNAP) molecule undergoes to achieve the initiation-elongation transition. Having made this transition, an RNAP molecule would be relinquished from its promoter hold to perform productive (full-length) transcription. Prior to the transition, this process is accompanied by abortive RNA formation—the amount and pattern of which is controlled by the promoter sequence information. Qualitative and quantitative analysis of abortive/productive transcription from several promoters and their sequence variants led to the understanding that a strong (RNAP-binding) promoter is more likely to be rate limited (during transcription initiation) at the escape step and produce abortive transcripts. Of the two subelements in a promoter, the PRR (the core Promoter Recognition Region) was found to set the initiation frequency and the rate-limiting step, while the ITS (the Initial Transcribed Sequence region) modulated the ratio of abortive versus productive transcription. The highly abortive behavior of RNAP could be ameliorated by the presence of Gre (transcript cleavage stimulatory) factor(s), linking the first step in abortive RNA formation by the initial transcribing complexes (ITC) to RNAP backtracking. The discovery that translocation during the initiation stage occurs via DNA scrunching provided the source of energy that converts each ITC into a highly unstable "stressed intermediate." Mapping all of the biochemical information onto an X-ray crystallographic structural model of an open complex gave rise to a plausible mechanism of transcription initiation. The chapter concludes with contemplations of the kinetics and thermodynamics of abortive initiation-promoter escape.

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Figures

Image of Figure 1
Figure 1

Shown are the products of transcription from six T5 -related promoter templates that differ only in their initial transcribed sequences (ITS). Compared with the native promoter, , , and differ in sequences from +3 to +20, and and differ only from +3 to +10. Each ITS directs the synthesis of a distinct collection of abortive RNAs whose sizes (in nucleotides) are indicated by numbers on the border. A set of five reactions (lanes a to e) was performed with each promoter to show that the abortive pattern is greatly altered by the presence of GreB (lanes c and e) and unaffected by NusA (lane d) compared with the normal pattern of abortive/productive synthesis (lanes a and b). FL designates the full-length productive RNA.

Citation: Hsu L. 2008. Promoter Escape by RNA Polymerase, EcoSal Plus 2008; doi:10.1128/ecosalplus.4.5.2.2
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Image of Figure 2
Figure 2

The promoters analyzed are , , and . Abortive probability at each initial position is derived from the quantitative measurement of abortive and productive RNA levels from a slice of gel shown below each panel; the abortive yield of an RNA is further corrected by the fraction of RNA polymerase reaching that position ( 38 ). High abortive probability reflects a highly unstable ITC that tends to release its RNA and begin initiation again. Plotting the abortive probability associated with each initial position produces the abortive probability profile that conveys the ease or difficulty of traversing the initial transcribed sequence region by RNA polymerase. The journey is different on each promoter.

Citation: Hsu L. 2008. Promoter Escape by RNA Polymerase, EcoSal Plus 2008; doi:10.1128/ecosalplus.4.5.2.2
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Image of Figure 3
Figure 3

At escape rate-limited promoters, transcription initiation follows a branched pathway due to the formation of two types of open complex. In this scheme, R represents RNA polymerase; P, promoter DNA; RP, the closed complex; RP, the productive open complex that can escape rounds of abortive cycling to synthesize full-length RNA; RP′, the unproductive open complex that abortively initiates without undergoing escape; and TEC, ternary elongation complex. The numbers 2, 3, 4, 5, and 6 represent initial transcribing complexes (ITCs) with a nascent RNA of the same length. is the equilibrium binding constant for closed complex formation; and are forward and reverse rate constants for open complex formation and collapse; is a composite rate constant of promoter escape that measures the rate of productive RNA synthesis from an open complex. An arrow underneath each ITC is of different length to represent the different levels of abortive RNA released.

Citation: Hsu L. 2008. Promoter Escape by RNA Polymerase, EcoSal Plus 2008; doi:10.1128/ecosalplus.4.5.2.2
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Figure 4

Thermodynamic considerations lead to two postulates. (A) Abortive potential predicts the occurrence of structural equilibration of ITCs at each initial position. Shown here are the ITC conformations associated with two successive steps of incorporation, for example, the seventh and eighth step in the transcription, where the seventh step is one of high abortive potential and the eighth step is one of low abortive potential. At the th initial position, at least four conformations of the ITC can be distinguished by their superscript designations: , the pretranslocation conformation; , the posttranslocation, scrunched conformation; , the backtracked conformation; and , the forward-incorporating conformation. ITC and ITC are related through the scrunching translocation mechanism, whereas ITC , ITC , and ITC are related through the structural equilibration steps to stabilize the ITC. ITC harbors high strain energy; it can be stabilized through one of two means: by backtracking to form ITC , which subsequently releases its RNA to resume the stable open complex structure (RP), or by propagating the strain energy away from the active site, forming a stable structure (ITC ) competent at incorporating the next nucleotide and moving the transcription complex forward. The lengths of the versus arrows are drawn to indicate its relationship with abortive probability. (B) The free-energy diagram of promoter escape at a promoter is related to its abortive probability profile. The example shown is that of the promoter. RP, open complex; EC, elongation complex. See text for discussion on the relationship of abortive probability to the free-energy minimum of an ITC.

Citation: Hsu L. 2008. Promoter Escape by RNA Polymerase, EcoSal Plus 2008; doi:10.1128/ecosalplus.4.5.2.2
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