Dominant lethal mutations near the 5' substrate binding site affect RNA polymerase propagation.

Sagitov, V; Nikiforov, Vadim; Goldfarb, Alex

doi:10.1016/s0021-9258(18)53981-8

Cited by 40 publications

(2 citation statements)

References 40 publications

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“…These complexes are not blocked at the nucleotide addition phase as evidenced by the synthesis of abortive transcripts up to 9-15 nt in length, but are unable to bring about the promoter escape transition to produce the full-length RNA. Consistent with this notion, a number of RNA polymerase mutants that are able to perform abortive initiation but unable to achieve promoter escape have previously been characterized (54)(55)(56). Alternatively, the promoter escape transition can be brought about sooner by RNA polymerase mutations in a specific region of σ 70 (57).…”

Section: Discussionmentioning

confidence: 87%

In Vitro Studies of Transcript Initiation by Escherichia coli RNA Polymerase. 2. Formation and Characterization of Two Distinct Classes of Initial Transcribing Complexes

Hsu

Kane

et al. 2003

Biochemistry

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By following the kinetics of abortive and productive synthesis in single-round transcription assays, we confirm the existence of two general classes of initial transcribing complexes (ITCs), which we term "productive ITC" and "unproductive ITC". The productive ITCs are able to escape from the promoter rapidly to produce full-length transcripts, but only after carrying out an obligate series of abortive initiation steps. The unproductive ITCs were found to synthesize mostly abortive transcripts of 2-3 nucleotides and escape from the promoter extremely slowly, if at all. Formation of the unproductive ITC is not due to the inactive RNA polymerase. Instead, RNA polymerase molecules recovered from both the productive and unproductive ITC fractions were shown to carry out abortive and productive synthesis with both the partitioning tendency and transcription kinetics similar to those of the original enzyme. Our results suggest that early transcription complexes are partitioned into the productive and unproductive ITCs most likely during the formation of open promoter complexes. The extent of partitioning varies with individual promoter sequences and is dependent on the nature and concentration of the initiating nucleotide. Thus, multiple classes of ITCs can be formed during promoter binding and transcript initiation.

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Section: Discussionmentioning

confidence: 87%

In Vitro Studies of Transcript Initiation by Escherichia coli RNA Polymerase. 2. Formation and Characterization of Two Distinct Classes of Initial Transcribing Complexes

Hsu

Kane

et al. 2003

Biochemistry

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“…Substituting alanine for lysl065 has only a minor effect on catalysis. Fikewise, mutants with a variety of single and multiple substi tutions between aa 1063 to 1073 still have significant ^ 15% transcriptional activity in multiple round transcription assays (Sagitov et al 1993). However, such mutants do exhibit altered transcriptional proper ties including weakened binding of nucleotides at both the initiation and elongation sites of the enzyme, and aberrant patterns of abortive initiation, promoter clearance and pausing.…”

Section: (A) L a N Dm Arks In The Tw O Largest Su Bun Itsmentioning

confidence: 99%

A structure/function analysis ofEscherichia coliRNA polymerase

Gross

Chan

Lonetto³

1996

Phil. Trans. R. Soc. Lond. B

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Control of RNA polymerase is a common means of regulating gene expression. A detailed picture of both the structure and how the structural details of RNA polymerase encode function is a key to understanding the molecular strategies used to regulate RNA polymerase. We review here data which ascribes functions to some regions of the primary sequence of the subunits (alpha, beta beta' sigma) which make up E. coli RNA polymerase. We review both genetic and biochemical data which place regions of the primary sequence that are distant from one another in close proximity in the tertiary structure. Finally we discuss the implications of these findings on the quaternary structure of RNA polymerase.

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A universally conserved region of the largest subunit participates in the active site of RNA polymerase III.

et al. 1995

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The largest subunits of the three eukaryotic nuclear RNA polymerase present extensive sequence homology with the beta' subunit of the bacterial enzymes over five major co‐linear regions. Region d is the most highly conserved and contains a motif, (Y/F)NADFDGD(E/Q)M(N/A), which is invariant in all multimeric RNA polymerases. An extensive mutagenesis of that region in yeast RNA polymerase III led to a vast majority (16/22) of lethal single‐site substitutions. A few conditional mutations were also obtained. One of them, rpc160–112, corresponds to a double substitution (T506I, N509Y) and has a slow growth phenotype at 25 degrees C. RNA polymerase III from the mutant rpc160–112 was severely impaired in its ability to transcribe a tRNA gene in vitro. The transcription defect did not originate from a deficiency in transcription complex formation and RNA chain initiation, but was mainly due to a reduced elongation rate. Under conditions of substrate limitation, the mutant enzyme showed increased pausing at the intrinsic pause sites of the SUP4 tRNA gene and an increased rate of slippage of nascent RNA, as compared with the wild‐type enzyme. The enzyme defect was also detectable with poly[d(A‐T)] as template, in the presence of saturating DNA, ATP and UTP concentrations. The mutant enzyme behavior is best explained by a distortion of the active site near the growing point of the RNA product.

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Dominant lethal mutations near the 5' substrate binding site affect RNA polymerase propagation.

Cited by 40 publications

References 40 publications

In Vitro Studies of Transcript Initiation by Escherichia coli RNA Polymerase. 2. Formation and Characterization of Two Distinct Classes of Initial Transcribing Complexes

In Vitro Studies of Transcript Initiation by Escherichia coli RNA Polymerase. 2. Formation and Characterization of Two Distinct Classes of Initial Transcribing Complexes

A structure/function analysis ofEscherichia coliRNA polymerase

A universally conserved region of the largest subunit participates in the active site of RNA polymerase III.

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