In Vitro Transcription and Start Site Selection in Schizosaccharomyces pombe

Choi, Wai; Yan, Ming; Nusinow, Dmitri A.; Gralla, Jay D.

doi:10.1016/s0022-2836(02)00329-7

Cited by 22 publications

(30 citation statements)

References 43 publications

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“…The transcription start site is located Ϸ30 bp downstream of the TATA box in S. pombe and humans, whereas it varies from 40 to 120 bp downstream in S. cerevisiae (19). TFIIB has been implicated in start site selection (20)(21)(22)(23), and human TFIIB can substitute for endogenous TFIIB in S. pombe, whereas S. cerevisiae TFIIB cannot (24). The Rpb4/Rpb7 dimer is stoichiometric in S. pombe and human.…”

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confidence: 99%

Schizosacharomyces pombe RNA polymerase II at 3.6-Å resolution

Spåhr

Calero

Bushnell

et al. 2009

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

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The second structure of a eukaryotic RNA polymerase II so far determined, that of the enzyme from the fission yeast Schizosaccharomyces pombe, is reported here. Comparison with the previous structure of the enzyme from the budding yeast Saccharomyces cerevisiae reveals differences in regions implicated in start site selection and transcription factor interaction. These aspects of the transcription mechanism differ between S. pombe and S. cerevisiae, but are conserved between S. pombe and humans. Amino acid changes apparently responsible for the structural differences are also conserved between S. pombe and humans, suggesting that the S. pombe structure may be a good surrogate for that of the human enzyme. molecular replacement ͉ X-ray crystallography ͉ yeast X -ray crystal structures of RNA polymerases from bacteria and Archaea, and of RNA polymerase II (pol II) from the budding yeast Saccharomyces cerevisiae, have given insight into the fundamental mechanism of transcription (1-7). The structures are closely similar in the catalytic core of the enzyme, where mobile elements interact with nucleic acids during transcription. The bacterial and S. cerevisiae structures differ in the periphery, where the enzymes interact with accessory factors, most notably the general transcription factors and Mediator of eukaryotes. One of the general factors, transcription factor II B (TFIIB), exhibits a degree of conservation with bacterial sigma factor and has a counterpart in Archaea, and a subunit of another general factor, the TATA-binding protein, occurs in Archaea as well, but the remaining general factors and Mediator are unique to eukaryotes.Conserved elements of the catalytic core include ''switch regions'' at the base of a swinging clamp, which interact with the DNA-RNA hybrid and downstream DNA during transcription (2, 3) and may be involved in transcription start site determination (8, 9). A so-called ''trigger loop'' interacts with the substrate nucleoside triphosphate to achieve the high fidelity of transcription (10)(11)(12)(13)(14). A ''bridge helix'' interacts with the coding base in the template strand of the DNA and is believed to play a role in the translocation step of transcription.Peripheral, more divergent elements of pol II include subunits Rpb4 and Rpb7, which form a dimer protruding from the enzyme surface (15, 16) and may be involved in RNA binding. Nearby lies the C terminus of Rpb1, from which extends a flexible linker, followed by Ͼ20 repeats of a 7-aa sequence [the C-terminal domain (CTD)], which interact not only with Mediator but also with capping, splicing, and cleavage/polyadenylation factors during transcription (17).Pol II is 53% identical in amino acid sequence between S. cerevisiae and humans, assuring a high degree of structural similarity. Notable differences in transcription have nevertheless emerged, including differences in promoter structure and interactions with accessory factors. In these respects, the fission yeast Schizosaccharomyces pombe more closely resembles the human system....

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confidence: 99%

Schizosacharomyces pombe RNA polymerase II at 3.6-Å resolution

Spåhr

Calero

Bushnell

et al. 2009

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

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“…We chose S. pombe as a genetically amenable organism for this purpose. Prior studies (35) have shown that transcription in this organism is much closer than S. cerevisiae to that of mammalian systems. An overexpression system exists in which TFIIB and other general transcription factor mutants have been characterized in S. pombe (34).…”

Section: Effects Of the B-tip Mutations In Vivo-mentioning

confidence: 93%

“…Cultures were grown to an A 595 of 0.8. Cells were harvested and processed to make noodles and whole cell transcription extracts as described (35).…”

Section: Proteins and Creation Of Transcription Complexes-tfiibmentioning

confidence: 99%

“…An overexpression system exists in which TFIIB and other general transcription factor mutants have been characterized in S. pombe (34). A study of the role of TFIIB in transcription initiation has suggested that S. pombe TFIIB has properties that better mimic those of human TFIIB (35). In addition, S. pombe extracts containing cloned general transcription factors have been shown to contain a complement of factors that faithfully direct RNA polymerase II transcription (34,41).…”

Section: Effects Of the B-tip Mutations In Vivo-mentioning

confidence: 99%

“…4A. Both wild type and D4 forms of S. pombe TFIIB were transformed into S. pombe FY191, and doxycycline was added to induce overexpression as described (35). The dominant negative system is known to result in overexpression of cloned TFIIB in a background containing the endogenous wild type TFIIB expressed from the chromosome.…”

Section: Effects Of the B-tip Mutations In Vivo-mentioning

confidence: 99%

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The TFIIB Tip Domain Couples Transcription Initiation to Events Involved in RNA Processing

Tran

Gralla

2010

Journal of Biological Chemistry

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TFIIB is the only factor within the multimegadalton transcription complex that is obligatorily required to undergo dissociation and re-association with each round of mRNA transcription. Here we show that a six-amino acid human TFIIB tip region is needed for appropriate levels of serine 5 C-terminal domain phosphorylation and mRNA capping and for retention of the required elongation factor TFIIF. We suggest that the broad functions of this tiny region are used to suppress transcription noise by restricting functional RNA synthesis from non-promoter sites on the genome, which will not contain TFIIB.To induce proper transcription by RNA polymerase II, on the order of 100 polypeptides assemble on DNA, directed by various promoter elements (1). The function of this giant complex is to recruit RNA polymerase to an appropriate site on the chromosome and to activate it so it can accomplish RNA synthesis. Only one of these polypeptides, TFIIB, must be obligatorily released from the template during this process for transcription to proceed. Thus, TFIIB plays a unique role in transcription initiation. Why TFIIB must associate and then be released and what this TFIIB cycle accomplishes is not well understood.TFIIB is known to both directly recruit RNA polymerase and to alter its enzymatic properties (2-4). The recruitment relies on several protein domains to bind components of the transcription preinitiation complex as well as to bind RNA polymerase. One of these domains is the "B-finger" or "Breader loop," which has a tip that approaches the active site of RNA polymerase within the preinitiation complex (5-7). This tip contains a pair of aspartate residues that bind magnesium and are required for full catalysis by RNA polymerase during the initiation phase of transcription (3-4). The assistance presumably provides a quality control function in that RNA polymerases that associate "non-specifically" with the genome, which is without the assistance of TFIIB and the associated preinitiation complex, will have low catalytic activity.TFIIB has a number of binding partners within the preinitiation complex in addition to RNA polymerase, TATA-binding protein, to direct it to promoter regions, the RNA polymerase elongation partner IIF, and many other factors including acidic activators (8). Despite these many stabilizing interactions, the release of TFIIB is believed to be required during each transcription cycle (9). This occurs during the process of escape when the polymerase is converted into a fully competent RNA synthesis machine.The physiological purpose of this release is not known but is presumably related to the many changes in the RNA polymerase and the associated machinery that is coupled to the escape process (10 -15). These changes set in motion a series of events that allow proper elongation of transcription and couple elongation to RNA processing (16). Central to this program is the phosphorylation of the C-terminal domain of the RNA polymerase. In humans the largest RNA polymerase II subunit has 52 repeats of a hepta...

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Cloning, sequencing and application of the LEU2 gene from the sour dough yeast Candida milleri

Turakainen

Korhola

2005

Yeast

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We have cloned by complementation in Saccharomyces cerevisiae and sequenced a LEU2 gene from the sour dough yeast Candida milleri CBS 8195 and studied its chromosomal location. The LEU2 coding sequence was 1092 nt long encoding a putative β-isopropylmalate dehydrogenase protein of 363 amino acids. The nucleotide sequence in the coding region had 71.6% identity to S. cerevisiae LEU2 sequence. On the protein level, the identity of C. milleri Leu2p to S. cerevisiae Leu2p was 84.1%. The DNA sequence of the C. milleri LEU2 gene was submitted to GenBank and assigned Accession No. AY571337. The CmLEU2 DNA probe hybridized to one to three chromosomal bands and two or three BamHI restriction fragments in C. milleri but did not give any signal to chromosomes or restriction fragments of C. albicans, S. cerevisiae, S. exiguus or Torulaspora delbrueckii. Using CmLEU2 probe for DNA hybridization makes it easy to quickly identify C. milleri among other sour dough yeasts.

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In Vitro Transcription and Start Site Selection in Schizosaccharomyces pombe

Cited by 22 publications

References 43 publications

Schizosacharomyces pombe RNA polymerase II at 3.6-Å resolution

Schizosacharomyces pombe RNA polymerase II at 3.6-Å resolution

The TFIIB Tip Domain Couples Transcription Initiation to Events Involved in RNA Processing

Cloning, sequencing and application of the LEU2 gene from the sour dough yeast Candida milleri

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