Engineering of Elongation Complexes of Bacterial and Yeast RNA Polymerases

Комиссарова, Н. В.; Kireeva, Maria L.; Becker, Jodi; Sidorenkov, I. V.; Kashlev, Mikhail

doi:10.1016/s0076-6879(03)71017-9

Cited by 82 publications

(90 citation statements)

References 24 publications

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“…To determine how Nun arrests elongating RNAP, we assembled a TEC in vitro from RNAP core enzyme carrying a His 6 tag in the C terminus of the β′ subunit, as well as synthetic TDS65, RNA9 and NDS65 (14,29) (Table S1). The template DNA sequence used in these studies was derived from the T7A1 promoter.…”

Section: Resultsmentioning

confidence: 99%

“…The TECs were assembled in transcription buffer (TB) [20 mM Tris·HCl (pH 7.9), 5 mM MgCl 2 , 40 mM KCl, 2 mM 2-mercaptoethanol], with 10-20 pmol of purified RNAP, 5′-labeled RNA primer annealed to the template DNA strand (TDS) and nontemplate DNA strand (NDS) subsequently added ( Fig. 1A) as previously described (14,29). The nomenclature of the TECs is as follows: Taking, for example, the TEC 9G 9 , the RNA length is indicated by the first number (9), the letter (G) identifies the 3′ residue of the RNA primer/ transcript, and the subscripted number ( 9 ) indicates the distance from the arbitrary +1A position in the nontemplate DNA to the 3′ end of the RNA (the transcription start site of the T7A1 promoter from which the oligonucleotide sequence was derived).…”

Section: Methodsmentioning

confidence: 99%

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Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Vitiello

Kireeva

Lubkowska

et al. 2014

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

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The Nun protein of coliphage HK022 arrests RNA polymerase (RNAP) in vivo and in vitro at pause sites distal to phage λ N-Utilization (nut) site RNA sequences. We tested the activity of Nun on ternary elongation complexes (TECs) assembled with templates lacking the λ nut sequence. We report that Nun stabilizes both translocation states of RNAP by restricting lateral movement of TEC along the DNA register. When Nun stabilized TEC in a pretranslocated register, immediately after NMP incorporation, it prevented binding of the next NTP and stimulated pyrophosphorolysis of the nascent transcript. In contrast, stabilization of TEC by Nun in a posttranslocated register allowed NTP binding and nucleotidyl transfer but inhibited pyrophosphorolysis and the next round of forward translocation. Nun binding to and action on the TEC requires a 9-bp RNA-DNA hybrid. We observed a Nun-dependent toe print upstream to the TEC. In addition, mutations in the RNAP β′ subunit near the upstream end of the transcription bubble suppress Nun binding and arrest. These results suggest that Nun interacts with RNAP near the 5′ edge of the RNA-DNA hybrid. By stabilizing translocation states through restriction of TEC lateral mobility, Nun represents a novel class of transcription arrest factors.T ranscription elongation is highly processive, yet the rate of nucleotide addition varies significantly for different ternary elongation complexes (TECs). In the normal elongation pathway, each nucleotide addition is followed by translocation of RNA polymerase (RNAP) along DNA in single-nucleotide increments. This process transfers the RNA 3′ end from the i + 1 site to the i site of the active center, thus allowing binding of the next NTP and subsequent phosphodiester bond formation. Translocation is thought to be a stochastic, rapid, and fully reversible process and not rate-limiting for elongation (1, 2). Instead, NTP sequestration, phosphodiester bond formation, or pyrophosphate release has been suggested to be rate-limiting for transcription elongation (3, 4). However, several reports strongly suggest that translocation may be at least partially rate-limiting for elongation (5-9).Immediately following bond formation, a catalytically inactive and highly pyrophosphorolytic (pretranslocated) state is formed. In this state, the 3′ RNA end remains in the i + 1 site of the active center. The 3′ RNA thus prevents NTP binding and generates a substrate for pyrophosphorolysis. To bind the next NTP, RNAP must translocate 1-bp forward along the DNA register to form a catalytically active and pyrophosphate-resistant (posttranslocated) state. Stationary RNAP has been suggested to "ratchet" between the two states via Brownian motion (5). NTP bound to the transient posttranslocated state acts as a pawl that interferes with backward translocation, thereby favoring the formation of a phosphodiester bond. Retention of this NTP in the posttranslocated TEC is facilitated by isomerization of the active site that blocks substrate exit, and aligns it for catalysis (10, 11)...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Vitiello

Kireeva

Lubkowska

et al. 2014

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

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“…Pol II Purification and RNA and DNA Oligonucleotides-Pol II carrying hexahistidine-tagged Rpb3 and E. coli RNA polymerase (RNAP) were purified as described (23,24). The DNA and RNA oligonucleotides for ternary elongation complex (TEC) assembly were from IDT (Coralville, IA): RNA9, 5Ј-AUC -nitrilotriacetic acid-agarose beads, TEC purification, and elution with imidazole were performed as described (23).…”

Section: Methodsmentioning

confidence: 99%

The Fidelity of Transcription

Strathern

Malagón

Irvin

et al. 2013

Journal of Biological Chemistry

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Background: Yeast RNA polymerase II domains involved in maintenance of transcription register are unknown. Results: We isolated and biochemically characterized RPB1 mutations leading to register loss (transcription slippage). Conclusion: All RPB1 mutations affect slippage localize close to the active center and near the secondary pore of RNA polymerase II. Significance: Our results shed light on the mechanism of slippage by eukaryotic RNA polymerases.

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“…We used a minimal in vitro transcription system involving ternary elongation complexes (TECs) reconstituted from RNAP core enzyme and synthetic RNA/DNA oligonucleotides (35). This system was used to test whether slippage on the T5C5 motif depends only on the interaction of RNAP with the transcript and template in the 9-to 10-bp RNA-DNA hybrid as has been shown for slippage on homopolymeric tracts (22,36).…”

Section: Resultsmentioning

confidence: 99%

“…Briefly, to prepare the initial TEC (Fig. 5A), the TEC was first assembled from synthetic RNA and DNA oligonucleotides (35) and then ligated to a DNA test insert. The TEC was walked by addition of NTP subsets to form a 63 nt transcript stalled 8 nt upstream of the slippery site (3′-A5G5-5′) on the DNA template.…”

Section: Methodsmentioning

confidence: 99%

Productive mRNA stem loop-mediated transcriptional slippage: Crucial features in common with intrinsic terminators

Penno

Sharma

Coakley

et al. 2015

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

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Escherichia coli and yeast DNA-dependent RNA polymerases are shown to mediate efficient nascent transcript stem loop formation-dependent RNA-DNA hybrid realignment. The realignment was discovered on the heteropolymeric sequence T5C5 and yields transcripts lacking a C residue within a corresponding U5C4. The sequence studied is derived from a Roseiflexus insertion sequence (IS) element where the resulting transcriptional slippage is required for transposase synthesis. The stability of the RNA structure, the proximity of the stem loop to the slippage site, the length and composition of the slippage site motif, and the identity of its 3′ adjacent nucleotides (nt) are crucial for transcripts lacking a single C. In many respects, the RNA structure requirements for this slippage resemble those for hairpin-dependent transcription termination. In a purified in vitro system, the slippage efficiency ranges from 5% to 75% depending on the concentration ratios of the nucleotides specified by the slippage sequence and the 3′ nt context. The only previous proposal of stem loop mediated slippage, which was in Ebola virus expression, was based on incorrect data interpretation. We propose a mechanical slippage model involving the RNAP translocation state as the main motor in slippage directionality and efficiency. It is distinct from previously described models, including the one proposed for paramyxovirus, where following random movement efficiency is mainly dependent on the stability of the new realigned hybrid. In broadening the scope for utilization of transcription slippage for gene expression, the stimulatory structure provides parallels with programmed ribosomal frameshifting at the translation level.transcriptional realignment | stem loop stimulator | heteropolymeric slippage-prone motifs | frameshifting A relatively neglected aspect of gene expression is functionally important RNA-DNA hybrid realignment within the RNA polymerase (RNAP) transcribing coding sequence. Such realigned polymerases yield transcripts lacking, or containing, one or more additional base(s) corresponding to the slippage-prone sequence. The fraction of the mRNA population containing deletions or insertions with respect to the template that are not of 3 nucleotides (nt) or multiples thereof, yields proteins that are either truncated, have C-terminal regions not encoded by the zero frame of the template DNA, or both. Despite the limited investigations, several diverse functional slippage-derived products have already been characterized.Bacterial multisubunit DNA-dependent RNAPs undergo slippage on homopolymeric runs of As or Ts. Transcriptional slippage on a run of nine As in the Thermus thermophilus dnaX gene results in heterogeneous transcripts that lead to 50% of the ribosomes quickly terminating. This termination yields a DNA polymerase subunit that lacks C-terminal domains and is synthesized in a 1:1 ratio with the full-length product (1). In other bacteria, counterpart slippage is required for synthesis of the fulllength rather than the truncat...

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Engineering of Elongation Complexes of Bacterial and Yeast RNA Polymerases

Cited by 82 publications

References 24 publications

Coliphage HK022 Nun protein inhibits RNA polymerase translocation

Coliphage HK022 Nun protein inhibits RNA polymerase translocation

The Fidelity of Transcription

Productive mRNA stem loop-mediated transcriptional slippage: Crucial features in common with intrinsic terminators

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