Termination and pausing of RNA polymerase II downstream of yeast polyadenylation sites (original) (raw)
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Transcriptional termination signals for RNA polymerase II in fission yeast
The EMBO journal, 1997
Transcription 'run-on' (TRO) analysis using permeabilized yeast cells indicates that transcription terminates between 180 and 380 bp downstream of the poly(A) site of the Schizosaccharomyces pombe ura4 gene. Two signals direct RNA polymerase II (pol II) to stop transcription: the ...
Budding yeast RNA polymerases I and II employ parallel mechanisms of transcriptional termination
Genes & Development, 2008
Both RNA polymerase I and II (Pol I and Pol II) in budding yeast employ a functionally homologous “torpedo-like” mechanism to promote transcriptional termination. For two well-defined Pol II-transcribed genes, CYC1 and PMA1, we demonstrate that both Rat1p exonuclease and Sen1p helicase are required for efficient termination by promoting degradation of the nascent transcript associated with Pol II, following mRNA 3′ end processing. Similarly, Pol I termination relies on prior Rnt1p cleavage at the 3′ end of the pre-rRNA 35S transcript. This is followed by the combined actions of Rat1p and Sen1p to degrade the Pol I-associated nascent transcript that consequently promote termination in the downstream rDNA spacer sequence. Our data suggest that the previously defined in vitro Pol I termination mechanism involving the action of the Reb1p DNA-binding factor to “road-block” Pol I transcription close to the termination region may have overlooked more complex in vivo molecular processes.
Disengaging polymerase: Terminating RNA polymerase II transcription in budding yeast
Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 2013
Termination of transcription by RNA polymerase II requires two distinct processes: The formation of a defined 3′ end of the transcribed RNA, as well as the disengagement of RNA polymerase from its DNA template. Both processes are intimately connected and equally pivotal in the process of functional messenger RNA production. However, research in recent years has elaborated how both processes can additionally be employed to control gene expression in qualitative and quantitative ways. This review embraces these new findings and attempts to paint a broader picture of how this final step in the transcription cycle is of critical importance to many aspects of gene regulation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.
G3 (Bethesda, Md.), 2013
Transcription termination by RNA polymerase (Pol) II is an essential but poorly understood process. In eukaryotic nuclei, the 3' ends of mRNAs are generated by cleavage and polyadenylation, and the same sequence elements that specify that process are required for downstream release of the polymerase from the DNA. Although Pol II is known to bind proteins required for both events, few studies have focused on Pol II mutations as a means to uncover the mechanisms that couple polyadenylation and termination. We performed a genetic screen in the yeast Saccharomyces cerevisiae to isolate mutations in the N-terminal half of Rpb2, the second largest Pol II subunit, that conferred either a decreased or increased response to a well-characterized poly(A) site. Most of the mutant alleles encoded substitutions affecting either surface residues or conserved active site amino acids at positions important for termination by other RNA polymerases. Reverse transcription polymerase chain reaction ...
Nucleic Acids Research, 2002
The poly(A) signal and downstream elements with transcriptional pausing activity play an important role in termination of RNA polymerase II transcription. We show that an intronic sequence derived from the plant seed protein gene (AmA1) specifically acts as a transcriptional terminator in the fission yeast, Schizosaccharomyces pombe. The 3′-end points of mRNA encoded by the AmA1 gene were mapped at different positions in S.pombe and in native cells of Amaranthus hypochondriacus. Deletion analyses of the AmA1 intronic sequence revealed that multiple elements essential for proper transcriptional termination in S.pombe include two site-determining elements (SDEs) and three downstream sequence elements. RT-PCR analyses detected transcripts up to the second SDE. This is the first report showing that the highly conserved mammalian poly(A) signal, AAUAAA, is also functional in S.pombe. The poly(A) site was determined as Y(A) both in native and heterologous systems but at different positions. Deletion of these cis-elements abolished 3′-end processing in S.pombe and a single point mutation in this motif reduced the activity by 70% while enhancing activity at downstream SDE. These results indicate that the bipartite sequence elements as signals for 3′-end processing in fission yeast act in tandem with other cis-acting elements. A comparison of these elements in the AmA1 intron that function as a transcriptional terminator in fission yeast with that of its native genes showed that both require an AT-rich distal and proximal upstream element. However, these sequences are not identical. Transcription run-on analysis indicates that elongating RNA polymerase II molecules accumulate over these pause signals, maximal at 611-949 nt. Furthermore, we demonstrate that the AmA1 intronic terminator sequence acts in a position-independent manner when placed within another gene.
In vivo analyses of RNA polymerase I termination in Schizosaccharomyces pombe
Nucleic Acids Research, 1997
Recent studies on the termination of rDNA transcription by RNA polymerase I in Saccharomyces cerevisiae and Schizosaccharomyces pombe have suggested a more complex mechanism then previously described in higher eukaryotes. Termination appears to occur when a DNA-bound Reb1 protein molecule induces polymerase to pause in the context of a release element [see Reeder,R.H. and Lang,W. (1994) Mol. Microbiol., 12, 11-15]. Because these conclusions in yeast were based entirely on in vitro analyses, we have examined the same termination process in S.pombe by expressing targeted mutations in vivo. S 1 nuclease protection studies indicate three tandemly arranged termination sites with most transcripts very efficiently terminated at the first site, 267 nt after the 3′ end of the mature 25S rRNA sequence. Termination at each site is mediated by conserved terminator elements which bear limited sequence homology with that of mouse and also can be identified in S.cerevisiae. Removal of the first terminator element transfers dominance to the second site and construction of a new single terminator element at +150 still results in efficient termination and rRNA processing without a need for an additional upstream element. Genomic 'footprint' analyses and gel retardation assays confirm a process mediated by a strongly interacting protein factor but implicate an alternate binding site. Removal of the 5′ flanking sequence or structure also had no effect on the site or efficiency of termination. Taken together the results in vivo suggest that the termination process in this fission yeast more strongly resembles the single element-mediated mechanism initially reported in mouse and is not dependent on additional upstream sequence as first reported in S.cerevisiae and postulated to function in general.
Nonpolyadenylated RNA Polymerase II Termination Is Induced by Transcript Cleavage
Journal of Biological Chemistry, 2008
Although the termination of transcription and 3 RNA processing of the eukaryotic mRNA has been linked to a polyadenylation signal and a transcript cleavage process, much less is known about the termination or processing of nonpolyadenylated RNA polymerase II transcripts. An efficiently expressed plasmid-based expression system was used to study the termination and processing of Schizosaccharomyces pombe U3 small nucleolar RNA (snoRNA) transcripts in vivo. The termination assay was linked to cell transformation, and restriction fragment length polymorphism was used to determine levels of plasmidderived U3 snoRNA. Mutation analyses in vivo indicate that the maturation of the 3 end is not directly dependent on an external cis-acting sequence or structure; rather, it is dependent on a transcript cleavage that can occur hundreds or even thousands of nucleotides downstream of the mature U3 snoRNA sequence. Similarly, termination is dependent on the same transcript cleavage that is localized in a hairpin structure that normally follows the 3 end of the U3 snoRNA but that also can be moved hundreds or thousands of nucleotides downstream. Both processes, however, can be induced simultaneously and equally efficiently with a single unrelated Pac1 endonuclease-labile structure. The results support a "reversed torpedoes" model in which a single cleavage allows exonucleases and/or other protein factors access to the transcript leading to transcription termination in one direction and RNA maturation in the other direction.
Transcription termination by nuclear RNA polymerases
2009
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.