The mechanism of transcription termination by RNA poiymerase I

Reeder, Ronald H.; Lang, Walter H.

doi:10.1111/j.1365-2958.1994.tb00989.x

Cited by 39 publications

(20 citation statements)

References 33 publications

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“…Furthermore, this orientation specificity is also not without precedent. Reb1, the yeast terminator for RNAP1, shares the Myb-like DNA binding domain and functions as a terminator in only the forward orientation with regard to transcription (Reeder and Lang 1994). Consistent with this model, we observed a large decrease in RNAP II occupancy downstream of the Rap1p site found within our CO-Ty1 element that did not occur when the Rap1p site was replaced with native Ty1 sequence.…”

Section: Discussionsupporting

confidence: 75%

“…Better known as the terminator for RNA polymerase I, the main role of Reb1p is to pause that polymerase at the release element, a role that can mostly be replaced by a heterologous DNA binding roadblock such as the lac repressor (Jeong et al 1995). Interestingly, like Rap1p, the DNA binding domain of Reb1p is similar to that of the Myb oncoprotein and likewise is only capable of pausing transcription in an orientation-specific manner (Reeder and Lang 1994). In humans, the transcription factor MAZ shares similarities with Rap1p, and is found to bind to promoters as well as to pause RNAP II transcription (Ashfield et al 1994;Yonaha and Proudfoot 1999).…”

Section: Discussionmentioning

confidence: 99%

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Novel Transcript Truncating Function of Rap1p Revealed by Synthetic Codon-Optimized Ty1 Retrotransposon

et al. 2012

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Extensive mutagenesis via massive recoding of retrotransposon Ty1 produced a synthetic codon-optimized retrotransposon (CO-Ty1). CO-Ty1 is defective for retrotransposition, suggesting a sequence capable of down-regulating retrotransposition. We mapped this sequence to a critical 20-bp region within CO-Ty1 reverse transcriptase (RT) and confirmed that it reduced Ty1 transposition, protein, and RNA levels. Repression was not Ty1 specific; when introduced immediately downstream of the green fluorescent protein (GFP) stop codon, GFP expression was similarly reduced. Rap1p mediated this down-regulation, as shown by mutagenesis and chromatin immunoprecipitation. A regular threefold drop is observed in different contexts, suggesting utility for synthetic circuits. A large reduction of RNAP II occupancy on the CO-Ty1 construct was observed 39 to the identified Rap1p site and a novel 39 truncated RNA species was observed. We propose a novel mechanism of transcriptional regulation by Rap1p whereby it serves as a transcriptional roadblock when bound to transcription unit sequences. SACCHAROMYCES cerevisiae Ty1 is the best-characterized and most abundant of five retrotransposon families present in the genome. Ty1 shares many similarities with retroviruses, such as HIV-1 and makes similar use of an RNA intermediate in its propagation. Unsurprisingly, this Ty1 RNA intermediate is essential for Ty1 retrotransposition. Not only does this "mRNA" code for the GAG and POL proteins required for retrotransposition, but it also serves a critical function as the genetic material for replication. Both Ty1 mRNA and its cDNA product contain cis-acting nucleotide determinants required for retrotransposition.Mutagenesis of Ty1 and screening for cis-acting nucleotide determinants has been previously performed with various strategies, including in-frame linker insertion mutagenesis Devine and Boeke 1994;Monokian et al. 1994), analysis of synthetic Ty1 DNA fragments to determine minimal requirements for the integration reaction (Eichinger and Boeke 1990;Braiterman et al. 1994;Devine and Boeke 1994;Sharon et al. 1994), deletion analysis and PCR-mediated mutagenesis of mini-Ty1 elements (Xu and Boeke 1990;Bolton et al. 2005), and comparative sequence analysis followed by targeted mutagenesis and/or generation of complementary mutations in RNA stem regions (Chapman et al. 1992;Heyman et al. 1995;Lauermann et al. 1995;Friant et al. 1998;Cristofari et al. 2002;Bolton et al. 2005). These studies provided insights into mechanisms underlying Ty1 retrotransposition and revealed several cis-acting sequences, including the Met i -tRNA:primer binding site (PBS) interaction (Chapman et al. 1992; Friant et al. 1998), LTRs (Eichinger andBraiterman et al. 1994;Devine and Boeke 1994;Sharon et al. 1994), polypurine tracts (PPTs) (Heyman et al. 1995;Lauermann et al. 1995), the GAG-POL frameshift region (Belcourt and Farabaugh 1990; Lawler et al. 2001), CYC5 and CYC3 (Cristofari et al. 2002), and an extensive 59 RNA structure required for efficient ini...

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Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Novel Transcript Truncating Function of Rap1p Revealed by Synthetic Codon-Optimized Ty1 Retrotransposon

et al. 2012

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“…The boxed sequence is predicted to span a membrane (16 (17,26). This observation is in keeping with the absence of homology between these terminators (26). As expected from the similarities of transcription between the procyclin, VSG, and ribosomal units (7), we found that the PARP A termination region was able to inhibit CAT activity driven by the VSG and ribosomal promoters, although this effect was reproducibly weaker in case of the VSG promoter.…”

Section: Discussionsupporting

confidence: 63%

“…S.L., splice leader acceptor site. The boxed sequence is predicted to span a membrane (16 (17,26). This observation is in keeping with the absence of homology between these terminators (26).…”

Section: Discussionsupporting

confidence: 54%

Characterization of a Transcription Terminator of the Procyclin PARP A Unit of Trypanosoma brucei

Berberof¹,

Pays²,

Lips³

et al. 1996

Molecular and Cellular Biology

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The polycistronic procyclin PARP (for procyclic acidic repetitive protein) A transcription unit of Trypanosoma brucei was completely characterized by the mapping of the termination region. In addition to the tandem of procyclin genes and GRESAG 2.1, this 7.5-to 9.5-kb unit contained another gene for a putative surface protein, termed PAG (for procyclin-associated gene) 3. The terminal 3-kb sequence did not contain significant open reading frames and cross-hybridized with the beginning of one or several transcription units specific to the bloodstream form. At least three separate fragments from the terminal region were able to inhibit chloramphenicol acetyltransferase expression when inserted between either the PARP, the ribosomal, or the variable surface glycoprotein promoter and a chloramphenicol acetyltransferase reporter gene. This inhibition was due to an orientation-dependent transcription termination caused by the combination of several attenuator elements with no obvious sequence conservation. The procyclin transcription terminator appeared unable to inhibit transcription by polymerase II.In Trypanosoma brucei, the genes seem to be generally organized in polycistronic transcription units (8,21). Because of this particular organization, only two trypanosomal promoters for protein-encoding genes are known, namely, those for the major stage-specific surface antigens, the variant surface glycoprotein (VSG) and procyclin (9,12,23,29,32). These promoters may recruit an RNA polymerase of the ribosomal type, although this remains equivocal (7,25). To date, no DNA sequence able to terminate transcription has been found in these organisms.Procyclin is the major surface glycoprotein of the procyclic form of T. brucei (27). The procyclin genes are organized in tandem pairs present in two diploid loci termed PARP (for procyclic acidic repetitive protein), A and PARP B, with the possible presence of an additional gene copy in some trypanosome strains (9,14,29). The transcription promoter is present immediately upstream from the first gene copy of each locus, and it seems that the different loci are transcribed simultaneously (9,14,29). In each locus, transcription is polycistronic, with at least one gene associated with the tandem of procyclin genes, GRESAG 2.1 in PARP A and PAG (for procyclinassociated gene) 1 in PARP B (2, 15). The approximative extent of each transcription unit was estimated to be less than 10 kb, contrasting with the larger size of units for other genes (2,9,15,25,29).In this paper, we report the complete characterization, from the promoter to the termination region, of the PARP A unit. There appear to be no more than four genes in this unit. Interestingly, the 3Ј-end region of the PARP A unit was found to cross-hybridize with the 5Ј-end region of at least one bloodstream-specific transcription unit. The termination region appeared to extend over 2 kb. Different fragments from this region efficiently inhibited chloramphenicol acetyltransferase (CAT) expression driven by either the PARP, the ribosom...

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“…How does the Reb1 TTD terminate transcription? Our data suggest an active mechanism promoted by physical interactions between Reb1 TTD and RNA pol I rather than a passive roadblock imposed by the tight binding of Reb1 to Ter DNA sites (7,47,48). To determine which subunit(s) of RNA pol I interacted with Reb1, we took advantage of the S. pombe genetic database (www.…”

Section: Resultsmentioning

confidence: 99%

Functional architecture of the Reb1-Ter complex of Schizosaccharomyces pombe

Jaiswal

Choudhury

Zaman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Reb1 of Schizosaccharomyces pombe represents a family of multifunctional proteins that bind to specific terminator sites (Ter) and cause polar termination of transcription catalyzed by RNA polymerase I (pol I) and arrest of replication forks approaching the Ter sites from the opposite direction. However, it remains to be investigated whether the same mechanism causes arrest of both DNA transactions. Here, we present the structure of Reb1 as a complex with a Ter site at a resolution of 2.7 Å. Structure-guided molecular genetic analyses revealed that it has distinct and well-defined DNA binding and transcription termination (TTD) domains. The region of the protein involved in replication termination is distinct from the TTD. Mechanistically, the data support the conclusion that transcription termination is not caused by just high affinity Reb1-Ter protein-DNA interactions. Rather, protein-protein interactions between the TTD with the Rpa12 subunit of RNA pol I seem to be an integral part of the mechanism. This conclusion is further supported by the observation that double mutations in TTD that abolished its interaction with Rpa12 also greatly reduced transcription termination thereby revealing a conduit for functional communications between RNA pol I and the terminator protein.crystal structure | RNA polymerase I | transcription termination | protein-DNA interaction | replication termination E ukaryotic rDNAs are found as multiple tandem copies encoding pre-rRNA and upstream and downstream regulatory elements (1-3) including DNA sequences (Ter) that promote site-specific termination of transcription catalyzed by RNA polymerase I (pol I) from yeast to humans (4-14). Specialized transcription terminator proteins bind to Ter sites and not only arrest transcription by RNA polymerase I (pol I) in a polar mode but also replication forks approaching from the opposite direction. Several studies have suggested that pol I transcription termination is a multistep process that requires (i) pausing of chain elongation by the terminator protein and (ii) dissociation and release of pol I and the primary transcript from the template. In mice, dissociation of pol I and release of the transcript require the release factor PTRF, a 44-kDa protein that interacts with the largest subunit of pol I (15). In yeast, additional factors for the processing of the end include the endonuclease Rnt1, the 5′-3′ Rat1 exonuclease, Sen1 helicase, and the kinase Grc3. They are part of an alternate pathway for termination by transcriptional coprocessing (4,(16)(17)(18)(19). In addition, in vivo analyses of Saccharomyces cerevisiae have shown a requirement for Rpa12, a component of pol I necessary for transcription termination (20). Whether the polar arrest of transcription is caused by an interaction between Rpa12 with the terminator protein is unknown.The terminator proteins that mediate transcription termination have been identified in multiple organisms and include Reb1 and Nsi1 [also called yeast transcription terminator (Ytt1)] of S. cerevisiae (...

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The mechanism of transcription termination by RNA poiymerase I

Cited by 39 publications

References 33 publications

Novel Transcript Truncating Function of Rap1p Revealed by Synthetic Codon-Optimized Ty1 Retrotransposon

Novel Transcript Truncating Function of Rap1p Revealed by Synthetic Codon-Optimized Ty1 Retrotransposon

Characterization of a Transcription Terminator of the Procyclin PARP A Unit of Trypanosoma brucei

Functional architecture of the Reb1-Ter complex of Schizosaccharomyces pombe

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