scholarly journals The primary σ factor in Escherichia coli can access the transcription elongation complex from solution in vivo

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Seth R Goldman ◽  
Nikhil U Nair ◽  
Christopher D Wells ◽  
Bryce E Nickels ◽  
Ann Hochschild
Keyword(s):  
Escherichia Coli ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Direct Effects ◽  
Elongation Complex ◽  
Initiation Factors ◽  
Bacterial Rna ◽  
Σ Factor ◽  
In Trans

The σ subunit of bacterial RNA polymerase (RNAP) confers on the enzyme the ability to initiate promoter-specific transcription. Although σ factors are generally classified as initiation factors, σ can also remain associated with, and modulate the behavior of, RNAP during elongation. Here we establish that the primary σ factor in Escherichia coli, σ70, can function as an elongation factor in vivo by loading directly onto the transcription elongation complex (TEC) in trans. We demonstrate that σ70 can bind in trans to TECs that emanate from either a σ70-dependent promoter or a promoter that is controlled by an alternative σ factor. We further demonstrate that binding of σ70 to the TEC in trans can have a particularly large impact on the dynamics of transcription elongation during stationary phase. Our findings establish a mechanism whereby the primary σ factor can exert direct effects on the composition of the entire transcriptome, not just that portion that is produced under the control of σ70-dependent promoters.

scholarly journals Role for the Ssu72 C-Terminal Domain Phosphatase in RNA Polymerase II Transcription Elongation

2006 ◽  
Vol 27 (3) ◽  
pp. 926-936 ◽  
Author(s):  
Mariela Reyes-Reyes ◽  
Michael Hampsey
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Elongation Complex ◽  
Terminal Domain ◽  
Rnap Ii ◽  
Transcription Cycle

ABSTRACT The RNA polymerase II (RNAP II) transcription cycle is accompanied by changes in the phosphorylation status of the C-terminal domain (CTD), a reiterated heptapeptide sequence (Y1S2P3T4S5P6S7) present at the C terminus of the largest RNAP II subunit. One of the enzymes involved in this process is Ssu72, a CTD phosphatase with specificity for serine-5-P. Here we report that the ssu72-2-encoded Ssu72-R129A protein is catalytically impaired in vitro and that the ssu72-2 mutant accumulates the serine-5-P form of RNAP II in vivo. An in vitro transcription system derived from the ssu72-2 mutant exhibits impaired elongation efficiency. Mutations in RPB1 and RPB2, the genes encoding the two largest subunits of RNAP II, were identified as suppressors of ssu72-2. The rpb1-1001 suppressor encodes an R1281A replacement, whereas rpb2-1001 encodes an R983G replacement. This information led us to identify the previously defined rpb2-4 and rpb2-10 alleles, which encode catalytically slow forms of RNAP II, as additional suppressors of ssu72-2. Furthermore, deletion of SPT4, which encodes a subunit of the Spt4-Spt5 early elongation complex, also suppresses ssu72-2, whereas the spt5-242 allele is suppressed by rpb2-1001. These results define Ssu72 as a transcription elongation factor. We propose a model in which Ssu72 catalyzes serine-5-P dephosphorylation subsequent to addition of the 7-methylguanosine cap on pre-mRNA in a manner that facilitates the RNAP II transition into the elongation stage of the transcription cycle.

scholarly journals Phosphorylation of the Transcription Elongation Factor Spt5 by Yeast Bur1 Kinase Stimulates Recruitment of the PAF Complex

2009 ◽  
Vol 29 (17) ◽  
pp. 4852-4863 ◽  
Author(s):  
Ying Liu ◽  
Linda Warfield ◽  
Chao Zhang ◽  
Jie Luo ◽  
Jasmina Allen ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Transcription Elongation ◽  
Chromatin Modification ◽  
Elongation Factor ◽  
Specific Inhibitor ◽  
Elongation Complex ◽  
Pol Ii ◽  
Paf Complex

ABSTRACT The Saccharomyces cerevisiae kinase Bur1 is involved in coupling transcription elongation to chromatin modification, but not all important Bur1 targets in the elongation complex are known. Using a chemical genetics strategy wherein Bur1 kinase was engineered to be regulated by a specific inhibitor, we found that Bur1 phosphorylates the Spt5 C-terminal repeat domain (CTD) both in vivo and in isolated elongation complexes in vitro. Deletion of the Spt5 CTD or mutation of the Spt5 serines targeted by Bur1 reduces recruitment of the PAF complex, which functions to recruit factors involved in chromatin modification and mRNA maturation to elongating polymerase II (Pol II). Deletion of the Spt5 CTD showed the same defect in PAF recruitment as rapid inhibition of Bur1 kinase activity, and this Spt5 mutation led to a decrease in histone H3K4 trimethylation. Brief inhibition of Bur1 kinase activity in vivo also led to a significant decrease in phosphorylation of the Pol II CTD at Ser-2, showing that Bur1 also contributes to Pol II Ser-2 phosphorylation. Genetic results suggest that Bur1 is essential for growth because it targets multiple factors that play distinct roles in transcription.

scholarly journals Analysis of Promoter Targets for Escherichia coli Transcription Elongation Factor GreA In Vivo and In Vitro

2007 ◽  
Vol 189 (24) ◽  
pp. 8772-8785 ◽  
Author(s):  
Ekaterina Stepanova ◽  
Jookyung Lee ◽  
Maria Ozerova ◽  
Ekaterina Semenova ◽  
Kirill Datsenko ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Expression Profiles ◽  
Transcription Elongation ◽  
Gene Array ◽  
Elongation Factor ◽  
Transcription Elongation Factor ◽  
Promoter Escape ◽  
Transcript Cleavage

ABSTRACT Transcription elongation factor GreA induces nucleolytic activity of bacterial RNA polymerase (RNAP). In vitro, transcript cleavage by GreA contributes to transcription efficiency by (i) suppressing pauses and arrests, (ii) stimulating RNAP promoter escape, and (iii) enhancing transcription fidelity. However, it is unclear which of these functions is (are) most relevant in vivo. By comparing global gene expression profiles of Escherichia coli strains lacking Gre factors and strains expressing either the wild type (wt) or a functionally inactive GreA mutant, we identified genes that are potential targets of GreA action. Data analysis revealed that in the presence of chromosomally expressed GreA, 19 genes are upregulated; an additional 105 genes are activated upon overexpression of the wt but not the mutant GreA. Primer extension reactions with selected transcription units confirmed the gene array data. The most prominent stimulatory effect (threefold to about sixfold) of GreA was observed for genes of ribosomal protein operons and the tna operon, suggesting that transcript cleavage by GreA contributes to optimal expression levels of these genes in vivo. In vitro transcription assays indicated that the stimulatory effect of GreA upon the transcription of these genes is mostly due to increased RNAP recycling due to facilitated promoter escape. We propose that transcript cleavage during early stages of initiation is thus the main in vivo function of GreA. Surprisingly, the presence of the wt GreA also led to the decreased transcription of many genes. The mechanism of this effect is unknown and may be indirect.

scholarly journals Probing the structure of Nun transcription arrest factor bound to RNA polymerase

2016 ◽  
Vol 113 (31) ◽  
pp. 8693-8698 ◽  
Author(s):  
Arkady Mustaev ◽  
Christal L. Vitiello ◽  
Max E. Gottesman
Keyword(s):  
Rna Polymerase ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
In Vitro Assays ◽  
Cross Linking ◽  
Elongation Complex ◽  
Rna:Dna Hybrid ◽  
Cross Links

The coliphage HK022 protein Nun transcription elongation arrest factor inhibits RNA polymerase translocation. In vivo, Nun acts specifically to block transcription of the coliphage λ chromosome. Using in vitro assays, we demonstrate that Nun cross-links RNA in an RNA:DNA hybrid within a ternary elongation complex (TEC). Both the 5′ and the 3′ ends of the RNA cross-link Nun, implying that Nun contacts RNA polymerase both at the upstream edge of the RNA:DNA hybrid and in the vicinity of the catalytic center. This finding suggests that Nun may inhibit translocation by more than one mechanism. Transcription elongation factor GreA efficiently blocked Nun cross-linking to the 3′ end of the transcript, whereas the highly homologous GreB factor did not. Surprisingly, both factors strongly suppressed Nun cross-linking to the 5′ end of the RNA, suggesting that GreA and GreB can enter the RNA exit channel as well as the secondary channel, where they are known to bind. These findings extend the known action mechanism for these ubiquitous cellular factors.

scholarly journals Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

2007 ◽  
Vol 27 (13) ◽  
pp. 4641-4651 ◽  
Author(s):  
Junjiang Fu ◽  
Ho-Geun Yoon ◽  
Jun Qin ◽  
Jiemin Wong
Keyword(s):  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Elongation Complex ◽  
Complex Activity ◽  
Interacting Protein ◽  
Pol Ii ◽  
Carboxy Terminal

ABSTRACT P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function.

scholarly journals Escherichia coli NusG links the lead ribosome with the transcription elongation complex

2019 ◽  
Author(s):  
Robert S. Washburn ◽  
Philipp K. Zuber ◽  
Ming Sun ◽  
Yaser Hashem ◽  
Bingxin Shen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Conclusive Evidence ◽  
Resonance Spectroscopy ◽  
Elongation Complex ◽  
Magnetic Resonance Spectroscopy Data ◽  
N Utilization ◽  
Reporter Assays ◽  
70S Ribosome ◽  
Functional Analyses

AbstractIt has been known for more than 50 years that transcription and translation are physically coupled in bacteria, but whether or not this coupling may be mediated by the two-domain protein N-utilization substance (Nus) G in Escherichia coli is still heavily debated. Here, we combine integrative structural biology and functional analyses to provide conclusive evidence that NusG can physically link transcription with translation by contacting both RNA polymerase and the ribosome. We present a cryo-electron microscopy structure of a NusG:70S ribosome complex and nuclear magnetic resonance spectroscopy data revealing simultaneous binding of NusG to RNAP and the intact 70S ribosome, providing the first direct structural evidence for NusG-mediated coupling. Furthermore, in vivo reporter assays show that recruitment of NusG occurs late in transcription and strongly depends on translation. Thus, our data suggest that coupling occurs initially via direct RNAP:ribosome contacts and is then mediated by NusG.

Crystal structure of the human transcription elongation factor DSIF hSpt4 subunit in complex with the hSpt5 dimerization interface

2009 ◽  
Vol 425 (2) ◽  
pp. 373-380 ◽  
Author(s):  
Sabine Wenzel ◽  
Berta M. Martins ◽  
Paul Rösch ◽  
Birgitta M. Wöhrl
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Structural Similarity ◽  
Transcription Elongation Factor

The eukaryotic transcription elongation factor DSIF [DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) sensitivity-inducing factor] is composed of two subunits, hSpt4 and hSpt5, which are homologous to the yeast factors Spt4 and Spt5. DSIF is involved in regulating the processivity of RNA polymerase II and plays an essential role in transcriptional activation of eukaryotes. At several eukaryotic promoters, DSIF, together with NELF (negative elongation factor), leads to promoter-proximal pausing of RNA polymerase II. In the present paper we describe the crystal structure of hSpt4 in complex with the dimerization region of hSpt5 (amino acids 176–273) at a resolution of 1.55 Å (1 Å=0.1 nm). The heterodimer shows high structural similarity to its homologue from Saccharomyces cerevisiae. Furthermore, hSpt5-NGN is structurally similar to the NTD (N-terminal domain) of the bacterial transcription factor NusG. A homologue for hSpt4 has not yet been found in bacteria. However, the archaeal transcription factor RpoE” appears to be distantly related. Although a comparison of the NusG-NTD of Escherichia coli with hSpt5 revealed a similarity of the three-dimensional structures, interaction of E. coli NusG-NTD with hSpt4 could not be observed by NMR titration experiments. A conserved glutamate residue, which was shown to be crucial for dimerization in yeast, is also involved in the human heterodimer, but is substituted for a glutamine residue in Escherichia coli NusG. However, exchanging the glutamine for glutamate proved not to be sufficient to induce hSpt4 binding.

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