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 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 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.

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

Purification and characterization of a transcription factor that confers promoter specificity to human RNA polymerase I

1985 ◽  
Vol 5 (6) ◽  
pp. 1358-1369
Author(s):  
R M Learned ◽  
S Cordes ◽  
R Tjian
Keyword(s):  
Rna Polymerase ◽  
In Vitro Transcription ◽  
Rna Polymerase I ◽  
Control Element ◽  
In Vitro Synthesis ◽  
Transcription System ◽  
Activation Of Transcription ◽  
Promoter Recognition ◽  
Polymerase I

A whole-cell HeLa extract was fractionated into two components required for accurate in vitro transcription of human rRNA. One fraction contained endogenous RNA polymerase I, and the second component contained a factor (SL1) that confers promoter selectivity to RNA polymerase I. Analysis of mutant templates suggests that the core control element of the rRNA promoter is required for activation of transcription by SL1. We purified SL1 approximately 100,000-fold by column chromatography and have shown that the addition of SL1 can reprogram the otherwise nonpermissive mouse transcription system to recognize and initiate accurate RNA synthesis from human rDNA. Antibodies raised against SL1 bind preferentially to a protein localized in the nucleolus of primate cells and specifically inhibit in vitro transcription initiating from the human rRNA promoter. By contrast, anti-SL1 does not react with the nucleolus of rodent cells and has no effect on the in vitro synthesis of mouse rRNA by a transcription system derived from mouse cells. These findings suggest that SL1 is a selectivity factor present in the nucleolus that imparts promoter recognition to RNA polymerase I and that can discriminate between rRNA promoters from different species.

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