scholarly journals A majority ofRhodobacter sphaeroidespromoters lack a crucial RNA polymerase recognition feature, enabling coordinated transcription activation

2020 ◽  
Vol 117 (47) ◽  
pp. 29658-29668
Author(s):  
Kemardo K. Henry ◽  
Wilma Ross ◽  
Kevin S. Myers ◽  
Kimberly C. Lemmer ◽  
Jessica M. Vera ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Stationary Phase ◽  
Bacterial Species ◽  
Bioinformatic Analysis ◽  
In Vitro Transcription ◽  
Rrna Synthesis ◽  
Transcription System ◽  
Bacterial Ribosome ◽  
Genome Wide

Using an in vitro transcription system with purified RNA polymerase (RNAP) to investigate rRNA synthesis in the photoheterotrophic α-proteobacteriumRhodobacter sphaeroides, we identified a surprising feature of promoters recognized by the major holoenzyme. Transcription fromR. sphaeroidesrRNA promoters was unexpectedly weak, correlating with absence of −7T, the very highly conserved thymine found at the last position in −10 elements of promoters in most bacterial species. Thymine substitutions for adenine at position −7 in the three rRNA promoters strongly increased intrinsic promoter activity, indicating thatR. sphaeroidesRNAP can utilize −7T when present. rRNA promoters were activated by purifiedR. sphaeroidesCarD, a transcription factor found in many bacterial species but not in β- and γ-proteobacteria. Overall, CarD increased the activity of 15 of 16 nativeR. sphaeroidespromoters tested in vitro that lacked −7T, whereas it had no effect on three of the four native promoters that contained −7T. Genome-wide bioinformatic analysis of promoters fromR. sphaeroidesand two other α-proteobacterial species indicated that 30 to 43% contained −7T, whereas 90 to 99% of promoters from non–α-proteobacteria contained −7T. Thus, promoters lacking −7T appear to be widespread in α-proteobacteria and may have evolved away from consensus to enable their coordinated regulation by transcription factors like CarD. We observed a strong reduction inR. sphaeroidesCarD levels when cells enter stationary phase, suggesting that reduced activation by CarD may contribute to inhibition of rRNA transcription when cells enter stationary phase, the stage of growth when bacterial ribosome synthesis declines.

The human RNA polymerase I structure reveals an HMG-like transcription factor docking domain specific to metazoans

2021 ◽  
Author(s):  
Julia L Daiß ◽  
Michael Pilsl ◽  
Kristina Straub ◽  
Andrea Bleckmann ◽  
Mona Höcherl ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
In Vitro Transcription ◽  
Cellular Growth ◽  
Hmg Box ◽  
Transcription Bubble ◽  
Transcription System ◽  
Pol I ◽  
Additional Domain

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP-fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This "dock II" domain resembles a truncated HMG-box incapable of DNA-binding which may serve as a downstream-transcription factor binding platform in metazoans. Biochemical analysis and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG-box domain containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

scholarly journals Genome-wide effects onEscherichia colitranscription from ppGpp binding to its two sites on RNA polymerase

2019 ◽  
Vol 116 (17) ◽  
pp. 8310-8319 ◽  
Author(s):  
Patricia Sanchez-Vazquez ◽  
Colin N. Dewey ◽  
Nicole Kitten ◽  
Wilma Ross ◽  
Richard L. Gourse
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Binding Sites ◽  
Promoter Sequence ◽  
In Vitro Transcription ◽  
Transcriptional Responses ◽  
Transcription Analysis ◽  
Genome Wide

The second messenger nucleotide ppGpp dramatically alters gene expression in bacteria to adjust cellular metabolism to nutrient availability. ppGpp binds to two sites on RNA polymerase (RNAP) inEscherichia coli, but it has also been reported to bind to many other proteins. To determine the role of the RNAP binding sites in the genome-wide effects of ppGpp on transcription, we used RNA-seq to analyze transcripts produced in response to elevated ppGpp levels in strains with/without the ppGpp binding sites on RNAP. We examined RNAs rapidly after ppGpp production without an accompanying nutrient starvation. This procedure enriched for direct effects of ppGpp on RNAP rather than for indirect effects on transcription resulting from starvation-induced changes in metabolism or on secondary events from the initial effects on RNAP. The transcriptional responses of all 757 genes identified after 5 minutes of ppGpp induction depended on ppGpp binding to RNAP. Most (>75%) were not reported in earlier studies. The regulated transcripts encode products involved not only in translation but also in many other cellular processes. In vitro transcription analysis of more than 100 promoters from the in vivo dataset identified a large collection of directly regulated promoters, unambiguously demonstrated that most effects of ppGpp on transcription in vivo were direct, and allowed comparison of DNA sequences from inhibited, activated, and unaffected promoter classes. Our analysis greatly expands our understanding of the breadth of the stringent response and suggests promoter sequence features that contribute to the specific effects of ppGpp.

scholarly journals A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH and recombinant TBP, TFIIB, TFIIE and TFIIF

2001 ◽  
Vol 268 (16) ◽  
pp. 4527-4536 ◽  
Author(s):  
Irina Kotova ◽  
Anna Lena Chabes ◽  
Bo Segerman ◽  
Sara Flodell ◽  
Lars Thelander ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
In Vitro Transcription ◽  
Transcription System

scholarly journals An integrated model for termination of RNA polymerase III transcription

2021 ◽  
Author(s):  
Juanjuan Xie ◽  
Umberto Aiello ◽  
Yves Clement ◽  
Nouhou Haidara ◽  
Mathias Girbig ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
In Vitro Transcription ◽  
Cis Acting ◽  
Template Strand ◽  
Polymerase Iii ◽  
Genome Wide ◽  
Fail Safe

RNA polymerase III (RNAPIII) synthesizes essential and abundant non-coding RNAs such as tRNAs. Controlling RNAPIII span of activity by accurate and efficient termination is a challenging necessity to ensure robust gene expression and to prevent conflicts with other DNA-associated machineries. The mechanism of RNAPIII termination is believed to be simpler than that of other eukaryotic RNA polymerases, solely relying on the recognition of a T-tract in the non-template strand. Here we combine high-resolution genome-wide analyses and in vitro transcription termination assays to revisit the mechanism of RNAPIII transcription termination in budding yeast. We show that T-tracts are necessary but not always sufficient for termination and that secondary structures of the nascent RNAs are important auxiliary cis-acting elements. Moreover, we show that the helicase Sen1 plays a key role in a fail-safe termination pathway. Our results provide a comprehensive model illustrating how multiple mechanisms cooperate to ensure efficient RNAPIII transcription termination.

scholarly journals In Vitro Transcription of pe38/Polyhedrin Hybrid Promoters Reveals Sequences Essential for Recognition by the Baculovirus-Induced RNA Polymerase and for the Strength of Very Late Viral Promoters

1998 ◽  
Vol 72 (4) ◽  
pp. 2991-2998 ◽  
Author(s):  
Ruud M. W. Mans ◽  
Dagmar Knebel-Mörsdorf
Keyword(s):  
Rna Polymerase ◽  
In Vitro Transcription ◽  
Sequence Motif ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Polyhedrin Promoter ◽  
Viral Promoters ◽  
Nuclear Polyhedrosis ◽  
Hybrid Promoters

ABSTRACT In vitro transcription was used to analyze the promoter specificity of the α-amanitin-resistant RNA polymerase that is induced late during infection of Autographa californica multicapsid nuclear polyhedrosis virus. By modifying the preparation of crude nuclear extracts, we have established an assay that permits differentiation between weak late and strong very late viral promoters. The virus-induced RNA polymerase initiates at a TAAG sequence motif in both late and very late promoters. Based on the sensitivity of our in vitro transcription system, we have investigated the sequences responsible for a functional TAAG motif and their putative role with respect to the strength of very late promoters. By constructing hybrid promoters between the early pe38 and the very late polyhedrin promoters, we demonstrated that the replacement of 7 nucleotides upstream of the nonfunctional TAAG sequences in the pe38 promoter with the corresponding sequences of the polyhedrin promoter was sufficient for recognition by the virus-induced RNA polymerase. The strength of the very late polyhedrin promoter was established after replacing the 5′ untranslated sequences of the pe38 promoter by those of the polyhedrin promoter in addition to the 7 nucleotides upstream of the TAAG motif.

Establishment of in vitro transcription system based on RNA-dependent RNA polymerase (RdRp) of Little cherry virus 1

2021 ◽  
Vol 65 (01) ◽  
pp. 101-103
Author(s):  
Xinran Cao ◽  
Yalan Wang ◽  
Guowei Geng ◽  
Yifan Sun ◽  
Chenmeng Gao ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
In Vitro Transcription ◽  
Rna Dependent Rna Polymerase ◽  
Transcription System
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