Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Mechanisms and Functions of the RNA Polymerase II General Transcription Machinery during the Transcription Cycle
Central to the development and survival of all organisms is the regulation of gene expression, which begins with the process of transcription catalyzed by RNA polymerases. During transcription of protein-coding genes, the general transcription factors (GTFs) work alongside RNA polymerase II (Pol II) to assemble the preinitiation complex at the transcription start site, open the promoter DNA, initiate synthesis of the nascent messenger RNA, transition to productive elongation, and ultimately terminate transcription. Through these different stages of transcription, Pol II is dynamically phosphorylated at the C-terminal tail of its largest subunit, serving as a control mechanism for Pol II elongation and a signaling/binding platform for co-transcriptional factors. The large number of core protein factors participating in the fundamental steps of transcription add dense layers of regulation that contribute to the complexity of temporal and spatial control of gene expression within any given cell type. The Pol II transcription system is highly conserved across different levels of eukaryotes; however, most of the information here will focus on the human Pol II system. This review walks through various stages of transcription, from preinitiation complex assembly to termination, highlighting the functions and mechanisms of the core machinery that participates in each stage.  more » « less
Award ID(s):
2242824
PAR ID:
10491092
Author(s) / Creator(s):
; ;
Publisher / Repository:
MDPI
Date Published:
Journal Name:
Biomolecules
Volume:
14
Issue:
2
ISSN:
2218-273X
Page Range / eLocation ID:
176
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; (, PLOS Pathogens)
    Daròs, José-Antonio (Ed.)
    Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription. 
    more » « less
  2. ; ; ; ; (, Proceedings of the National Academy of Sciences)
    Transcription must be properly regulated to ensure dynamic gene expression underlying growth, development, and response to environmental cues. Regulation is imposed throughout the transcription cycle, and while many efforts have detailed the regulation of transcription initiation and early elongation, the termination phase of transcription also plays critical roles in regulating gene expression. Transcription termination can be driven by only a few proteins in each domain of life. Detailing the mechanism(s) employed provides insight into the vulnerabilities of transcription elongation complexes (TECs) that permit regulated termination to control expression of many genes and operons. Here, we describe the biochemical activities and crystal structure of the superfamily 2 helicase Eta, one of two known factors capable of disrupting archaeal transcription elongation complexes. Eta retains a twin-translocase core domain common to all superfamily 2 helicases and a well-conserved C terminus wherein individual amino acid substitutions can critically abrogate termination activities. Eta variants that perturb ATPase, helicase, single-stranded DNA and double-stranded DNA translocase and termination activities identify key regions of the C terminus of Eta that, when combined with modeling Eta–TEC interactions, provide a structural model of Eta-mediated termination guided in part by structures of Mfd and the bacterial TEC. The susceptibility of TECs to disruption by termination factors that target the upstream surface of RNA polymerase and potentially drive termination through forward translocation and allosteric mechanisms that favor opening of the clamp to release the encapsulated nucleic acids emerges as a common feature of transcription termination mechanisms. 
    more » « less
  3. ; ; (, eLife Sciences Publications)
    Spatial organization of transcription machinery is emerging as a key regulator of gene expression, yet how RNA polymerases are organized at gene clusters remains unclear. Here, we show that RNA polymerases II and polymerase III form distinct nuclear foci at the 5S ribosomal DNA (rDNA)–Spliced leader 1 (SL1) cluster in C. elegans. Within this cluster, polymerase II binds to the SL1 gene, while polymerase III associates with 5S rDNA. Both polymerase foci display dynamic but distinct behaviors within the nucleus. The assembly of these polymerase foci is regulated across the cell cycle. ATTF-6, an AT-hook transcription factor, is essential for polymerases II foci formation but dispensable for polymerases III foci. While Pol III foci are largely resistant to temperature changes, Pol II foci are temperature-sensitive, and their dissolution correlates with reduced SL1 expression. Together, these results reveal a spatial and temporal regulation of two RNA polymerases that organize gene cluster transcription. 
    more » « less
  4. ; ; ; ; ; ; (, Nature Communications)
    Abstract Transcription-coupled repair (TCR) is a vital nucleotide excision repair sub-pathway that removes DNA lesions from actively transcribed DNA strands. Binding of CSB to lesion-stalled RNA Polymerase II (Pol II) initiates TCR by triggering the recruitment of downstream repair factors. Yet it remains unknown how transcription factor IIH (TFIIH) is recruited to the intact TCR complex. Combining existing structural data with AlphaFold predictions, we build an integrative model of the initial TFIIH-bound TCR complex. We show how TFIIH can be first recruited in an open repair-inhibited conformation, which requires subsequent CAK module removal and conformational closure to process damaged DNA. In our model, CSB, CSA, UVSSA, elongation factor 1 (ELOF1), and specific Pol II and UVSSA-bound ubiquitin moieties come together to provide interaction interfaces needed for TFIIH recruitment. STK19 acts as a linchpin of the assembly, orienting the incoming TFIIH and bridging Pol II to core TCR factors and DNA. Molecular simulations of the TCR-associated CRL4CSAubiquitin ligase complex unveil the interplay of segmental DDB1 flexibility, continuous Cullin4A flexibility, and the key role of ELOF1 for Pol II ubiquitination that enables TCR. Collectively, these findings elucidate the coordinated assembly of repair proteins in early TCR. 
    more » « less
  5. ; ; ; (, bioRxiv)
    Abstract Bacteria use a multi-layered regulatory strategy to precisely and rapidly tune gene expression in response to environmental cues. Small RNAs (sRNAs) form an important layer of gene expression control and most act post-transcriptionally to control translation and stability of mRNAs. We have shown that at least five different sRNAs inEscherichia coliregulate the cyclopropane fatty acid synthase (cfa) mRNA. These sRNAs bind at different sites in the long 5’ untranslated region (UTR) ofcfamRNA and previous work suggested that they modulate RNase E-dependent mRNA turnover. Recently, thecfa5’ UTR was identified as a site of Rho-dependent transcription termination, leading us to hypothesize that the sRNAs might also regulatecfatranscription elongation. In this study we find that a pyrimidine-rich region flanked by sRNA binding sites in thecfa5’ UTR is required for premature Rho-dependent termination. We discovered that both the activating sRNA RydC and repressing sRNA CpxQ regulatecfaprimarily by modulating Rho-dependent termination ofcfatranscription, with only a minor effect on RNase E-mediated turnover ofcfamRNA. A stem-loop structure in thecfa5’ UTR sequesters the pyrimidine-rich region required for Rho-dependent termination. CpxQ binding to the 5’ portion of the stem increases Rho-dependent termination whereas RydC binding downstream of the stem decreases termination. These results reveal the versatile mechanisms sRNAs use to regulate target gene expression at transcriptional and post-transcriptional levels and demonstrate that regulation by sRNAs in long UTRs can involve modulation of transcription elongation. ImportanceBacteria respond to stress by rapidly regulating gene expression. Regulation can occur through control of messenger RNA (mRNA) production (transcription elongation), stability of mRNAs, or translation of mRNAs. Bacteria can use small RNAs (sRNAs) to regulate gene expression at each of these steps, but we often do not understand how this works at a molecular level. In this study, we find that sRNAs inEscherichia coliregulate gene expression at the level of transcription elongation by promoting or inhibiting transcription termination by a protein called Rho. These results help us understand new molecular mechanisms of gene expression regulation in bacteria. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Text Encoded Conversion
  • XML
  • Save / Share this Record
  • Send to Email