Abstract:We identify Rpa12p of RNA polymerase I (Pol I) as a termination factor. Combined analyses using transcription run-on, electron microscopy-visualized chromatin spreading and RT-PCR have been applied to the rRNA-encoding genes of Saccharomyces cerevisiae. These confirm that Pol I termination occurs close to the Reb1p-dependent terminator in wild-type strains. However, deletion mutants for the 3 end-processing enzyme Rnt1p or the Rpa12p subunit of Pol I both show Pol I transcription in the spacer. For ⌬rpa12, the… Show more
“…While C11 is implicated in RNAPIII reinitiation (see below), Rpb9 might regulate arrest of elongating RNAPII at pause sites (Awrey et al 1997). TRO analysis and electron microscopy (EM) visualization of Miller spreads demonstrated the involvement of Rpa12 in RNAPI termination (Prescott et al 2004;Kawauchi et al 2008). These experiments link transcription termination and processing of pre-rRNA.…”
Section: Coupling Of Rrna Processing and Terminationmentioning
confidence: 90%
“…Inactivation of Rnt1 causes read-through at T1, indicating that, as with protein-coding genes, cotranscriptional cleavage is important for efficient termination Prescott et al 2004;Kawauchi et al 2008). Recently, Catala et al (2008) showed that Rnt1 interacts with two RNAPIspecific subunits, Rpa12 and Rpa34.…”
Section: Coupling Of Rrna Processing and Terminationmentioning
confidence: 99%
“…Ten percent of yeast RNAPI transcripts do not terminate at T1 and read-through to a second terminator (T2 or ''fail-safe'' terminator) located ;250 nt downsteam from the 25S RNA 39-end Prescott et al 2004) or to the replication fork barrier (RFB). The RFB, located roughly 300 bp downstream, recruits the protein Fob1, which blocks DNA replication forks (see Fig.…”
Gene transcription in the cell nucleus is a complex and highly regulated process. Transcription in eukaryotes requires three distinct RNA polymerases, each of which employs its own mechanisms for initiation, elongation, and termination. Termination mechanisms vary considerably, ranging from relatively simple to exceptionally complex. In this review, we describe the present state of knowledge on how each of the three RNA polymerases terminates and how mechanisms are conserved, or vary, from yeast to human.
“…While C11 is implicated in RNAPIII reinitiation (see below), Rpb9 might regulate arrest of elongating RNAPII at pause sites (Awrey et al 1997). TRO analysis and electron microscopy (EM) visualization of Miller spreads demonstrated the involvement of Rpa12 in RNAPI termination (Prescott et al 2004;Kawauchi et al 2008). These experiments link transcription termination and processing of pre-rRNA.…”
Section: Coupling Of Rrna Processing and Terminationmentioning
confidence: 90%
“…Inactivation of Rnt1 causes read-through at T1, indicating that, as with protein-coding genes, cotranscriptional cleavage is important for efficient termination Prescott et al 2004;Kawauchi et al 2008). Recently, Catala et al (2008) showed that Rnt1 interacts with two RNAPIspecific subunits, Rpa12 and Rpa34.…”
Section: Coupling Of Rrna Processing and Terminationmentioning
confidence: 99%
“…Ten percent of yeast RNAPI transcripts do not terminate at T1 and read-through to a second terminator (T2 or ''fail-safe'' terminator) located ;250 nt downsteam from the 25S RNA 39-end Prescott et al 2004) or to the replication fork barrier (RFB). The RFB, located roughly 300 bp downstream, recruits the protein Fob1, which blocks DNA replication forks (see Fig.…”
Gene transcription in the cell nucleus is a complex and highly regulated process. Transcription in eukaryotes requires three distinct RNA polymerases, each of which employs its own mechanisms for initiation, elongation, and termination. Termination mechanisms vary considerably, ranging from relatively simple to exceptionally complex. In this review, we describe the present state of knowledge on how each of the three RNA polymerases terminates and how mechanisms are conserved, or vary, from yeast to human.
Transcription of ribosomal RNA by RNA polymerase (Pol) I initiates ribosome biogenesis and regulates eukaryotic cell growth. The crystal structure of Pol I fromthe yeast Saccharomyces cerevisiae at 2.8A˚ resolution reveals all 14 subunits of the 590-kilodalton enzyme, and shows differences to Pol II. An ‘expander’ element occupies the DNA template site and stabilizes an expanded active centre cleft with an unwound bridge helix. A ‘connector’ element invades the cleft of an adjacent polymerase and stabilizes an inactive polymerase dimer. The connector and expander must detach during Pol I activation to enable transcription initiation and cleft contraction by convergent movement of the polymerase ‘core’ and ‘shelf’ modules. Conversion between an inactive expanded and an active contracted polymerase state may generally underlie transcription. Regulatory factors can modulate the core–shelf interface that includes a ‘composite’ active site for RNA chain initiation, elongation, proofreading and termination
“…Rpa190 was depleted in a tet:: regulated allele; Rpa12 encodes a nonessential subunit of Pol I involved in transcription termination and was directly obliterated in the genome of haploid yeast cells. Logarithmic yeast cultures were treated with 10% sarkosyl, which permeabilizes cell membranes and reversibly blocks elongating polymerases (Prescott et al 2004), incubated with [a 32 P]-UTP and transcription allowed to resume for the optimized labeling time of 5 min. Neosynthesized, radiolabeled transcripts were extracted, partially hydrolyzed, and used to probe slot-blots loaded with singlestranded DNA fragments complementary to the entire rDNA locus (Fig.…”
Section: Christmas Tree Visualization In Vivo By Ch-ipmentioning
Terminal balls detected at the 59-end of nascent ribosomal transcripts act as pre-rRNA processing complexes and are detected in all eukaryotes examined, resulting in illustrious Christmas tree images. Terminal balls (also known as SSU-processomes) compaction reflects the various stages of cotranscriptional ribosome assembly. Here, we have followed SSU-processome compaction in vivo by use of a chromatin immunoprecipitation (Ch-IP) approach and shown, in agreement with electron microscopy analysis of Christmas trees, that it progressively condenses to come in close proximity to the 59-end of the 25S rRNA gene. The SSU-processome is comprised of independent autonomous building blocks that are loaded onto nascent pre-rRNAs and assemble into catalytically active pre-rRNA processing complexes in a stepwise and highly hierarchical process. Failure to assemble SSU-processome subcomplexes with proper kinetics triggers a nucleolar surveillance pathway that targets misassembled pre-rRNAs otherwise destined to mature into small subunit 18S rRNA for polyadenylation, preferentially by TRAMP5, and degradation by the 39 to 59 exoribonucleolytic activity of the Exosome. Trf5 colocalized with nascent pre-rRNPs, indicating that this nucleolar surveillance initiates cotranscriptionally.
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