scholarly journals Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Sarah K Stumper ◽  
Harini Ravi ◽  
Larry J Friedman ◽  
Rachel Anne Mooney ◽  
Ivan R Corrêa ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Transcription Initiation ◽  
Inhibition Mechanism ◽  
Sufficient Time ◽  
Secondary Channel ◽  
Rna Transcription ◽  
Conformational Rearrangement ◽  
Rrna Transcription ◽  
Promoter Complex

RNA polymerases (RNAPs) contain a conserved ‘secondary channel’ which binds regulatory factors that modulate transcription initiation. In Escherichia coli, the secondary channel factors (SCFs) GreB and DksA both repress ribosomal RNA (rRNA) transcription, but SCF loading and repression mechanisms are unclear. We observed in vitro fluorescently labeled GreB molecules binding to single RNAPs and initiation of individual transcripts from an rRNA promoter. GreB arrived and departed from promoters only in complex with RNAP. GreB did not alter initial RNAP-promoter binding but instead blocked a step after conformational rearrangement of the initial RNAP-promoter complex. Strikingly, GreB-RNAP complexes never initiated at an rRNA promoter; only RNAP molecules arriving at the promoter without bound GreB produced transcript. The data reveal that a model SCF functions by a ‘delayed inhibition’ mechanism and suggest that rRNA promoters are inhibited by GreB/DksA because their short-lived RNAP complexes do not allow sufficient time for SCFs to dissociate.

Events during eucaryotic rRNA transcription initiation and elongation: conversion from the closed to the open promoter complex requires nucleotide substrates

1988 ◽  
Vol 8 (5) ◽  
pp. 1940-1946
Author(s):  
E Bateman ◽  
M R Paule
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Topological Analysis ◽  
Acanthamoeba Castellanii ◽  
Rna Polymerase I ◽  
Initiation Factor ◽  
Rrna Transcription ◽  
Promoter Complex ◽  
Polymerase I

Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyrocarbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.

scholarly journals Author response: Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription

2019 ◽  
Author(s):  
Sarah K Stumper ◽  
Harini Ravi ◽  
Larry J Friedman ◽  
Rachel Anne Mooney ◽  
Ivan R Corrêa ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Author Response ◽  
Inhibition Mechanism ◽  
Secondary Channel ◽  
Rna Transcription

scholarly journals Events during eucaryotic rRNA transcription initiation and elongation: conversion from the closed to the open promoter complex requires nucleotide substrates.

1988 ◽  
Vol 8 (5) ◽  
pp. 1940-1946 ◽  
Author(s):  
E Bateman ◽  
M R Paule
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Topological Analysis ◽  
Acanthamoeba Castellanii ◽  
Rna Polymerase I ◽  
Initiation Factor ◽  
Rrna Transcription ◽  
Promoter Complex ◽  
Polymerase I

Chemical footprinting and topological analysis were carried out on the Acanthamoeba castellanii rRNA transcription initiation factor (TIF) and RNA polymerase I complexes with DNA during transcription initiation and elongation. The results show that the binding of TIF and polymerase to the promoter does not alter the supercoiling of the DNA template and the template does not become sensitive to modification by diethylpyrocarbonate, which can identify melted DNA regions. Thus, in contrast to bacterial RNA polymerase, the eucaryotic RNA polymerase I-promoter complex is in a closed configuration preceding addition of nucleotides in vitro. Initiation and 3'-O-methyl CTP-limited translocation by RNA polymerase I results in separation of the polymerase-TIF footprints, leaving the TIF footprint unaltered. In contrast, initiation and translocation result in a significant change in the conformation of the polymerase-DNA complex, culminating in an unwound DNA region of at least 10 base pairs.

scholarly journals TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp

2017 ◽  
Vol 114 (28) ◽  
pp. E5539-E5548 ◽  
Author(s):  
Saumya Gopalkrishnan ◽  
Wilma Ross ◽  
Albert Y. Chen ◽  
Richard L. Gourse
Keyword(s):  
Active Site ◽  
Transcription Initiation ◽  
Coiled Coil ◽  
Global Regulator ◽  
Secondary Channel ◽  
Global Regulators ◽  
Extrachromosomal Elements ◽  
Distant Homolog ◽  
Active Site Region

TheEscherichia coliF element-encoded protein TraR is a distant homolog of the chromosome-encoded transcription factor DksA. Here we address the mechanism by which TraR acts as a global regulator, inhibiting some promoters and activating others. We show that TraR regulates transcription directly in vitro by binding to the secondary channel of RNA polymerase (RNAP) using interactions similar, but not identical, to those of DksA. Even though it binds to RNAP with only slightly higher affinity than DksA and is only half the size of DksA, TraR by itself inhibits transcription as strongly as DksA and ppGpp combined and much more than DksA alone. Furthermore, unlike DksA, TraR activates transcription even in the absence of ppGpp. TraR lacks the residues that interact with ppGpp in DksA, and TraR binding to RNAP uses the residues in the β′ rim helices that contribute to the ppGpp binding site in the DksA–ppGpp–RNAP complex. Thus, unlike DksA, TraR does not bind ppGpp. We propose a model in which TraR mimics the effects of DksA and ppGpp together by binding directly to the region of the RNAP secondary channel that otherwise binds ppGpp, and its N-terminal region, like the coiled-coil tip of DksA, engages the active-site region of the enzyme and affects transcription allosterically. These data provide insights into the function not only of TraR but also of an evolutionarily widespread and diverse family of TraR-like proteins encoded by bacteria, as well as bacteriophages and other extrachromosomal elements.

scholarly journals Structural basis for the function of SuhB as a transcription factor in ribosomal RNA synthesis

2019 ◽  
Vol 47 (12) ◽  
pp. 6488-6503 ◽  
Author(s):  
Yong-Heng Huang ◽  
Nelly Said ◽  
Bernhard Loll ◽  
Markus C Wahl
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Rna Folding ◽  
In Vitro Transcription ◽  
Structural Basis ◽  
Macromolecular Crystallography ◽  
Rna Transcription ◽  
Signal Element

AbstractRibosomal RNA synthesis in Escherichia coli involves a transcription complex, in which RNA polymerase is modified by a signal element on the transcript, Nus factors A, B, E and G, ribosomal protein S4 and inositol mono-phosphatase SuhB. This complex is resistant to ρ-dependent termination and facilitates ribosomal RNA folding, maturation and subunit assembly. The functional contributions of SuhB and their structural bases are presently unclear. We show that SuhB directly binds the RNA signal element and the C-terminal AR2 domain of NusA, and we delineate the atomic basis of the latter interaction by macromolecular crystallography. SuhB recruitment to a ribosomal RNA transcription complex depends on the RNA signal element but not on the NusA AR2 domain. SuhB in turn is required for stable integration of the NusB/E dimer into the complex. In vitro transcription assays revealed that SuhB is crucial for delaying or suppressing ρ-dependent termination, that SuhB also can reduce intrinsic termination, and that SuhB-AR2 contacts contribute to these effects. Together, our results reveal functions of SuhB during ribosomal RNA synthesis and delineate some of the underlying molecular interactions.

scholarly journals Transcription initiation of Xenopus 5S ribosomal RNA genes in vitro

1979 ◽  
Vol 7 (4) ◽  
pp. 947-958 ◽  
Author(s):  
Laurence J. Korn ◽  
Edward H. Birkenmeier ◽  
Donald D. Brown
Keyword(s):  
Ribosomal Rna ◽  
Transcription Initiation ◽  
Ribosomal Rna Genes ◽  
5S Ribosomal Rna ◽  
Rna Genes

Ribosomal RNA transcription in vitro is species specific

Nature ◽  
1982 ◽  
Vol 296 (5853) ◽  
pp. 173-174 ◽  
Author(s):  
Ingrid Grummt ◽  
Erika Roth ◽  
Marvin R. Paule
Keyword(s):  
Ribosomal Rna ◽  
Rna Transcription ◽  
Transcription In Vitro ◽  
Species Specific
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