In VitroStudies of Transcript Initiation byEscherichia coliRNA Polymerase. 3. Influences of Individual DNA Elements within the Promoter Recognition Region on Abortive Initiation and Promoter Escape†

Biochemistry ◽  
2003 ◽  
Vol 42 (13) ◽  
pp. 3798-3811 ◽  
Author(s):  
Nam V. Vo ◽  
Lilian M. Hsu ◽  
Caroline M. Kane ◽  
Michael J. Chamberlin
Keyword(s):  
Promoter Escape ◽  
Promoter Recognition ◽  
Dna Elements ◽  
Transcript Initiation ◽  
Abortive Initiation

scholarly journals Probing the Transcription Mechanisms of Reovirus Cores with Molecules That Alter RNA Duplex Stability

2009 ◽  
Vol 83 (11) ◽  
pp. 5659-5670 ◽  
Author(s):  
Alexander A. Demidenko ◽  
Max L. Nibert
Keyword(s):  
Small Molecules ◽  
Dimethyl Sulfoxide ◽  
Active Fraction ◽  
Core Particle ◽  
Double Stranded Rna ◽  
Promoter Escape ◽  
Duplex Stability ◽  
Rna Duplexes ◽  
Dsrna Viruses ◽  
Transcript Initiation

ABSTRACT The mammalian reovirus (MRV) genome comprises 10 double-stranded RNA (dsRNA) segments, packaged along with transcriptase complexes inside each core particle. Effects of four small molecules on transcription by MRV cores were studied for this report, chosen for their known capacities to alter RNA duplex stability. Spermidine and spermine, which enhance duplex stability, inhibited transcription, whereas dimethyl sulfoxide and trimethylglycine, which attenuate duplex stability, stimulated transcription. Different mechanisms were identified for inhibition or activation by these molecules. With spermidine, one round of transcription occurred normally, but subsequent rounds were inhibited. Thus, inhibition occurred at the transition between the end of elongation in one round and initiation in the next round of transcription. Dimethyl sulfoxide or trimethylglycine, on the other hand, had no effect on transcription by a constitutively active fraction of cores in each preparation but activated transcription in another fraction that was otherwise silent for the production of elongated transcripts. Activation of this other fraction occurred at the transition between transcript initiation and elongation, i.e., at promoter escape. These results suggest that the relative stability of RNA duplexes is most important for certain steps in the particle-associated transcription cycles of dsRNA viruses and that small molecules are useful tools for probing these and probably other steps.

scholarly journals Structures and mechanism of transcription initiation by bacterial ECF factors

2019 ◽  
Vol 47 (13) ◽  
pp. 7094-7104 ◽  
Author(s):  
Chengli Fang ◽  
Lingting Li ◽  
Liqiang Shen ◽  
Jing Shi ◽  
Sheng Wang ◽  
...  
Keyword(s):  
Active Center ◽  
Transcription Initiation ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Specific Gene ◽  
Initiation Complex ◽  
E Coli ◽  
Promoter Escape ◽  
Promoter Recognition ◽  
Transcription Initiation Complex

Abstract Bacterial RNA polymerase (RNAP) forms distinct holoenzymes with extra-cytoplasmic function (ECF) σ factors to initiate specific gene expression programs. In this study, we report a cryo-EM structure at 4.0 Å of Escherichia coli transcription initiation complex comprising σE—the most-studied bacterial ECF σ factor (Ec σE-RPo), and a crystal structure at 3.1 Å of Mycobacterium tuberculosis transcription initiation complex with a chimeric σH/E (Mtb σH/E-RPo). The structure of Ec σE-RPo reveals key interactions essential for assembly of E. coli σE-RNAP holoenzyme and for promoter recognition and unwinding by E. coli σE. Moreover, both structures show that the non-conserved linkers (σ2/σ4 linker) of the two ECF σ factors are inserted into the active-center cleft and exit through the RNA-exit channel. We performed secondary-structure prediction of 27,670 ECF σ factors and find that their non-conserved linkers probably reach into and exit from RNAP active-center cleft in a similar manner. Further biochemical results suggest that such σ2/σ4 linker plays an important role in RPo formation, abortive production and promoter escape during ECF σ factors-mediated transcription initiation.

scholarly journals Translocation after Synthesis of a Four-Nucleotide RNA Commits RNA Polymerase II to Promoter Escape

2002 ◽  
Vol 22 (3) ◽  
pp. 762-773 ◽  
Author(s):  
Jennifer F. Kugel ◽  
James A. Goodrich
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Site ◽  
Molecular Mechanisms ◽  
Sequence Specificity ◽  
Promoter Escape ◽  
General Transcription Factors ◽  
Dna Elements ◽  
Promoter Dna ◽  
Transcription Complexes

ABSTRACT Transcription is a complex process, the regulation of which is crucial for cellular and organismic growth and development. Deciphering the molecular mechanisms that define transcription is essential to understanding the regulation of RNA synthesis. Here we describe the molecular mechanism of escape commitment, a critical step in early RNA polymerase II transcription. During escape commitment ternary transcribing complexes become stable and committed to proceeding forward through promoter escape and the remainder of the transcription reaction. We found that the point in the transcription reaction at which escape commitment occurs depends on the length of the transcript RNA (4 nucleotides [nt]) as opposed to the position of the active site of the polymerase with respect to promoter DNA elements. We found that single-stranded nucleic acids can inhibit escape commitment, and we identified oligonucleotides that are potent inhibitors of this specific step. These inhibitors bind RNA polymerase II with low nanomolar affinity and sequence specificity, and they block both promoter-dependent and promoter-independent transcription, the latter occurring in the absence of general transcription factors. We demonstrate that escape commitment involves translocation of the RNA polymerase II active site between synthesis of the third and fourth phosphodiester bonds. We propose that a conformational change in ternary transcription complexes occurs during translocation after synthesis of a 4-nt RNA to render complexes escape committed.

scholarly journals Direct Observation of Abortive Initiation and Promoter Escape within Single Immobilized Transcription Complexes

2006 ◽  
Vol 90 (4) ◽  
pp. 1419-1431 ◽  
Author(s):  
Emmanuel Margeat ◽  
Achillefs N. Kapanidis ◽  
Philip Tinnefeld ◽  
You Wang ◽  
Jayanta Mukhopadhyay ◽  
...  
Keyword(s):  
Direct Observation ◽  
Promoter Escape ◽  
Transcription Complexes ◽  
Abortive Initiation

scholarly journals Structural basis of ribosomal RNA transcription regulation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yeonoh Shin ◽  
M. Zuhaib Qayyum ◽  
Danil Pupov ◽  
Daria Esyunina ◽  
Andrey Kulbachinskiy ◽  
...  
Keyword(s):  
Complex Formation ◽  
Ribosomal Rna ◽  
Structural Basis ◽  
Α Subunit ◽  
Promoter Escape ◽  
Cryo Electron Microscopy ◽  
Promoter Recognition ◽  
Carboxyl Terminal ◽  
Open Complex Formation ◽  
Template Dna

AbstractRibosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. Here, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ70 holoenzyme during rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element using dimerized α subunit carboxyl-terminal domains and scrunches the template DNA with the σ finger and β’ lid to select the transcription start site favorable for rapid promoter escape. Promoter binding induces conformational change of σ domain 2 that opens a gate for DNA loading and ejects σ1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding also opens the DNA loading gate, which is not coupled to σ1.1 ejection and impedes open complex formation. These results provide a molecular basis for the exceptionally active rRNA transcription and its vulnerability to DksA/ppGpp.

scholarly journals Distinct Roles for the Helicases of TFIIH in Transcript Initiation and Promoter Escape

2000 ◽  
Vol 275 (4) ◽  
pp. 2532-2538 ◽  
Author(s):  
John Bradsher ◽  
Frederic Coin ◽  
Jean-Marc Egly
Keyword(s):  
Promoter Escape ◽  
Transcript Initiation

scholarly journals Structural basis of the complete poxvirus transcription initiation process

2021 ◽  
Author(s):  
Utz Fischer ◽  
Clemens Grimm ◽  
Julia Bartuli ◽  
Bettina Böttcher ◽  
Aladar Szalay
Keyword(s):  
Transcription Initiation ◽  
Structural Basis ◽  
Major Groove ◽  
Promoter Escape ◽  
Universal Principles ◽  
Upstream Promoter ◽  
Promoter Recognition ◽  
Infected Cells ◽  
Early Transcription ◽  
Shed Light

Abstract Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I likely fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription.

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