scholarly journals Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly

2015 ◽  
Vol 36 (5) ◽  
pp. 820-831 ◽  
Author(s):  
Jürgen Niesser ◽  
Felix R. Wagner ◽  
Dirk Kostrewa ◽  
Wolfgang Mühlbacher ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Protein Complexes ◽  
Hydrophobic Pocket ◽  
Pol Ii ◽  
Content Type ◽  
Open Conformation ◽  
Peptide Release ◽  
Hydrophobic Peptide

Biogenesis of the 12-subunit RNA polymerase II (Pol II) transcription complex requires so-called GPN-loop GTPases, but the function of these enzymes is unknown. Here we report the first crystal structure of a eukaryotic GPN-loop GTPase, theSaccharomyces cerevisiaeenzyme Npa3 (a homolog of human GPN1, also called RPAP4, XAB1, and MBDin), and analyze its catalytic mechanism. The enzyme was trapped in a GDP-bound closed conformation and in a novel GTP analog-bound open conformation displaying a conserved hydrophobic pocket distant from the active site. We show that Npa3 has chaperone activity and interacts with hydrophobic peptide regions of Pol II subunits that form interfaces in the assembled Pol II complex. Biochemical results are consistent with a model that the hydrophobic pocket binds peptides and that this can allosterically stimulate GTPase activity and subsequent peptide release. These results suggest that GPN-loop GTPases are assembly chaperones for Pol II and other protein complexes.

scholarly journals Different phosphoisoforms of RNA polymerase II engage the Rtt103 termination factor in a structurally analogous manner

2017 ◽  
Vol 114 (20) ◽  
pp. E3944-E3953 ◽  
Author(s):  
Corey M. Nemec ◽  
Fan Yang ◽  
Joshua M. Gilmore ◽  
Corinna Hintermair ◽  
Yi-Hsuan Ho ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Protein Complexes ◽  
Transcription Termination ◽  
Structural Data ◽  
Side Chain ◽  
Protein Coding ◽  
Pol Ii ◽  
Carboxyl Terminal ◽  
Nucleolar Rna

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcription. Signature phosphorylation patterns of Y1S2P3T4S5P6S7 heptapeptide repeats of the CTD engage specific “readers.” Whereas phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we identify a role for phospho-Thr4 in transcription termination at noncoding small nucleolar RNA (snoRNA) genes. Quantitative proteomics reveals an interactome of known readers as well as protein complexes that were not known to rely on Thr4 for association with Pol II. The data indicate a key role for Thr4 in engaging the machinery used for transcription elongation and termination. We focus on Rtt103, a protein that binds phospho-Ser2 and phospho-Thr4 marks and facilitates transcription termination at protein-coding genes. To elucidate how Rtt103 engages two distinct CTD modifications that are differentially enriched at noncoding genes, we relied on NMR analysis of Rtt103 in complex with phospho-Thr4– or phospho-Ser2–bearing CTD peptides. The structural data reveal that Rtt103 interacts with phospho-Thr4 in a manner analogous to its interaction with phospho-Ser2–modified CTD. The same set of hydrogen bonds involving either the oxygen on phospho-Thr4 and the hydroxyl on Ser2, or the phosphate on Ser2 and the Thr4 hydroxyl, can be formed by rotation of an arginine side chain, leaving the intermolecular interface otherwise unperturbed. This economy of design enables Rtt103 to engage Pol II at distinct sets of genes with differentially enriched CTD marks.

scholarly journals A role for the RNA pol II–associated PAF complex in AID-induced immune diversification

2012 ◽  
Vol 209 (11) ◽  
pp. 2099-2111 ◽  
Author(s):  
Katharina L. Willmann ◽  
Sara Milosevic ◽  
Siim Pauklin ◽  
Kerstin-Maike Schmitz ◽  
Gopinath Rangam ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Protein Complexes ◽  
Chromatin Modification ◽  
Cytosine Deamination ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Antibody Diversification ◽  
Dna Instability ◽  
Paf Complex

Antibody diversification requires the DNA deaminase AID to induce DNA instability at immunoglobulin (Ig) loci upon B cell stimulation. For efficient cytosine deamination, AID requires single-stranded DNA and needs to gain access to Ig loci, with RNA pol II transcription possibly providing both aspects. To understand these mechanisms, we isolated and characterized endogenous AID-containing protein complexes from the chromatin of diversifying B cells. The majority of proteins associated with AID belonged to RNA polymerase II elongation and chromatin modification complexes. Besides the two core polymerase subunits, members of the PAF complex, SUPT5H, SUPT6H, and FACT complex associated with AID. We show that AID associates with RNA polymerase-associated factor 1 (PAF1) through its N-terminal domain, that depletion of PAF complex members inhibits AID-induced immune diversification, and that the PAF complex can serve as a binding platform for AID on chromatin. A model is emerging of how RNA polymerase II elongation and pausing induce and resolve AID lesions.

scholarly journals Emerging Views on the CTD Code

2012 ◽  
Vol 2012 ◽  
pp. 1-19 ◽  
Author(s):  
David W. Zhang ◽  
Juan B. Rodríguez-Molina ◽  
Joshua R. Tietjen ◽  
Corey M. Nemec ◽  
Aseem Z. Ansari
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Regulation ◽  
Chromatin Remodeling ◽  
Posttranslational Modifications ◽  
Rna Processing ◽  
Transcription Initiation ◽  
Protein Complexes ◽  
Pol Ii

The C-terminal domain (CTD) of RNA polymerase II (Pol II) consists of conserved heptapeptide repeats that function as a binding platform for different protein complexes involved in transcription, RNA processing, export, and chromatin remodeling. The CTD repeats are subject to sequential waves of posttranslational modifications during specific stages of the transcription cycle. These patterned modifications have led to the postulation of the “CTD code” hypothesis, where stage-specific patterns define a spatiotemporal code that is recognized by the appropriate interacting partners. Here, we highlight the role of CTD modifications in directing transcription initiation, elongation, and termination. We examine the major readers, writers, and erasers of the CTD code and examine the relevance of describing patterns of posttranslational modifications as a “code.” Finally, we discuss major questions regarding the function of the newly discovered CTD modifications and the fundamental insights into transcription regulation that will necessarily emerge upon addressing those challenges.

scholarly journals Stress Induces Changes in the Phosphorylation of Trypanosoma cruzi RNA Polymerase II, Affecting Its Association with Chromatin and RNA Processing

2014 ◽  
Vol 13 (7) ◽  
pp. 855-865 ◽  
Author(s):  
Antônio Augusto Rocha ◽  
Nilmar Silvio Moretti ◽  
Sergio Schenkman
Keyword(s):  
Heat Shock ◽  
Trypanosoma Cruzi ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Transcription Control ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Content Type ◽  
Polycistronic Transcription

ABSTRACT The phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of RNA polymerase II (Pol II) controls several transcription-related events in eukaryotes. Trypanosomatids lack these typical repeats and display an unusual transcription control. RNA Pol II associates with the transcription site of the spliced leader (SL) RNA, which is used in the trans -splicing of all mRNAs transcribed on long polycistronic units. We found that Trypanosoma cruzi RNA Pol II associated with chromatin is highly phosphorylated. When transcription is inhibited by actinomycin D, the enzyme runs off from SL genes, remaining hyperphosphorylated and associated with polycistronic transcription units. Upon heat shock, the enzyme is dephosphorylated and remains associated with the chromatin. Transcription is partially inhibited with the accumulation of housekeeping precursor mRNAs, except for heat shock genes. DNA damage caused dephosphorylation and transcription arrest, with RNA Pol II dissociating from chromatin although staying at the SL. In the presence of calyculin A, the hyperphosphorylated form detached from chromatin, including the SL loci. These results indicate that in trypanosomes, the unusual RNA Pol II is phosphorylated during the transcription of SL and polycistronic operons. Different types of stresses modify its phosphorylation state, affecting pre-RNA processing.

scholarly journals The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III

2014 ◽  
Vol 14 (3) ◽  
pp. 216-227 ◽  
Author(s):  
Norma E. Padilla-Mejía ◽  
Luis E. Florencio-Martínez ◽  
Rodrigo Moreno-Campos ◽  
Juan C. Vizuet-de-Rueda ◽  
Ana M. Cevallos ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Leishmania Major ◽  
Single Gene ◽  
Rna Polymerase Iii ◽  
Transcriptional Analysis ◽  
Pol Ii ◽  
Content Type ◽  
Polymerase Iii ◽  
Pol Iii

ABSTRACT Eukaryotic tRNAs, transcribed by RNA polymerase III (Pol III), contain boxes A and B as internal promoter elements. One exception is the selenocysteine (Sec) tRNA (tRNA-Sec), whose transcription is directed by an internal box B and three extragenic sequences in vertebrates. Here we report on the transcriptional analysis of the tRNA-Sec gene in the protozoan parasite Leishmania major . This organism has unusual mechanisms of gene expression, including Pol II polycistronic transcription and maturation of mRNAs by trans splicing, a process that attaches a 39-nucleotide miniexon to the 5′ end of all the mRNAs. In L. major , tRNA-Sec is encoded by a single gene inserted into a Pol II polycistronic unit, in contrast to most tRNAs, which are clustered at the boundaries of polycistronic units. 5′ rapid amplification of cDNA ends and reverse transcription-PCR experiments showed that some tRNA-Sec transcripts contain the miniexon at the 5′ end and a poly(A) tail at the 3′ end, indicating that the tRNA-Sec gene is polycistronically transcribed by Pol II and processed by trans splicing and polyadenylation, as was recently reported for the tRNA-Sec genes in the related parasite Trypanosoma brucei . However, nuclear run-on assays with RNA polymerase inhibitors and with cells that were previously UV irradiated showed that the tRNA-Sec gene in L. major is also transcribed by Pol III. Thus, our results indicate that RNA polymerase specificity in Leishmania is not absolute in vivo , as has recently been found in other eukaryotes.

scholarly journals An mRNA Capping Enzyme Targets FACT to the Active Gene To Enhance the Engagement of RNA Polymerase II into Transcriptional Elongation

2017 ◽  
Vol 37 (13) ◽  
Author(s):  
Rwik Sen ◽  
Amala Kaja ◽  
Jannatul Ferdoush ◽  
Shweta Lahudkar ◽  
Priyanka Barman ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Elongation ◽  
Regulatory Pathway ◽  
Pol Ii ◽  
Content Type ◽  
Active Gene ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Pol Ii Pausing

ABSTRACT We have recently demonstrated that an mRNA capping enzyme, Cet1, impairs promoter-proximal accumulation/pausing of RNA polymerase II (Pol II) independently of its capping activity in Saccharomyces cerevisiae to control transcription. However, it is still unknown how Pol II pausing is regulated by Cet1. Here, we show that Cet1's N-terminal domain (NTD) promotes the recruitment of FACT (facilitates chromatin transcription that enhances the engagement of Pol II into transcriptional elongation) to the coding sequence of an active gene, ADH1, independently of mRNA-capping activity. Absence of Cet1's NTD decreases FACT targeting to ADH1 and consequently reduces the engagement of Pol II in transcriptional elongation, leading to promoter-proximal accumulation of Pol II. Similar results were also observed at other genes. Consistently, Cet1 interacts with FACT. Collectively, our results support the notion that Cet1's NTD promotes FACT targeting to the active gene independently of mRNA-capping activity in facilitating Pol II's engagement in transcriptional elongation, thus deciphering a novel regulatory pathway of gene expression.

scholarly journals Ccr4-Not and TFIIS Function Cooperatively To Rescue Arrested RNA Polymerase II

2015 ◽  
Vol 35 (11) ◽  
pp. 1915-1925 ◽  
Author(s):  
Arnob Dutta ◽  
Vinod Babbarwal ◽  
Jianhua Fu ◽  
Deborah Brunke-Reese ◽  
Diane M. Libert ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Elongation Factors ◽  
Genetic Studies ◽  
Content Type ◽  
Mrna Metabolism ◽  
N Terminus ◽  
Biochemical Analyses

Expression of the genome requires RNA polymerase II (RNAPII) to transcribe across many natural and unnatural barriers, and this transcription across barriers is facilitated by protein complexes called elongation factors (EFs). Genetic studies inSaccharomyces cerevisiaeyeast suggest that multiple EFs collaborate to assist RNAPII in completing the transcription of genes, but the molecular mechanisms of how they cooperate to promote elongation are not well understood. The Ccr4-Not complex participates in multiple steps of mRNA metabolism and has recently been shown to be an EF. Here we describe how Ccr4-Not and TFIIS cooperate to stimulate elongation. We find that Ccr4-Not and TFIIS mutations show synthetically enhanced phenotypes, and biochemical analyses indicate that Ccr4-Not and TFIIS work synergistically to reactivate arrested RNAPII. Ccr4-Not increases the recruitment of TFIIS into elongation complexes and enhances the cleavage of the displaced transcript in backtracked RNAPII. This is mediated by an interaction between Ccr4-Not and the N terminus of TFIIS. In addition to revealing insights into how these two elongation factors cooperate to promote RNAPII elongation, our study extends the growing body of evidence suggesting that the N terminus of TFIIS acts as a docking/interacting site that allows it to synergize with other EFs to promote RNAPII transcription.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

scholarly journals Structure of the p53/RNA polymerase II assembly

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shu-Hao Liou ◽  
Sameer K. Singh ◽  
Robert H. Singer ◽  
Robert A. Coleman ◽  
Wei-Li Liu
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Conformational Changes ◽  
P53 Protein ◽  
Binding Activity ◽  
Transactivation Domain ◽  
Pol Ii ◽  
Dna Binding Activity ◽  
Regulated Gene Expression

AbstractThe tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

scholarly journals RecQL5 Promotes Genome Stabilization through Two Parallel Mechanisms—Interacting with RNA Polymerase II and Acting as a Helicase

2010 ◽  
Vol 30 (10) ◽  
pp. 2460-2472 ◽  
Author(s):  
M. Nurul Islam ◽  
David Fox ◽  
Rong Guo ◽  
Takemi Enomoto ◽  
Weidong Wang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genome Stability ◽  
Transcriptional Regulators ◽  
Helicase Activity ◽  
Parallel Mechanisms ◽  
Recq Helicase ◽  
Pol Ii ◽  
Kix Domain ◽  
Genome Stabilization

ABSTRACT The RecQL5 helicase is essential for maintaining genome stability and reducing cancer risk. To elucidate its mechanism of action, we purified a RecQL5-associated complex and identified its major component as RNA polymerase II (Pol II). Bioinformatics and structural modeling-guided mutagenesis revealed two conserved regions in RecQL5 as KIX and SRI domains, already known in transcriptional regulators for Pol II. The RecQL5-KIX domain binds both initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase, whereas the RecQL5-SRI domain interacts only with the elongation form. Fully functional RecQL5 requires both helicase activity and associations with the initiation polymerase, because mutants lacking either activity are partially defective in the suppression of sister chromatid exchange and resistance to camptothecin-induced DNA damage, and mutants lacking both activities are completely defective. We propose that RecQL5 promotes genome stabilization through two parallel mechanisms: by participation in homologous recombination-dependent DNA repair as a RecQ helicase and by regulating the initiation of Pol II to reduce transcription-associated replication impairment and recombination.

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