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Regulation of TATA-binding protein dynamics in living yeast cells.

ABSTRACT: Although pathways for assembly of RNA polymerase (Pol) II transcription preinitiation complexes (PICs) have been well established in vitro, relatively little is known about the dynamic behavior of Pol II general transcription factors in vivo. In vitro, a subset of Pol II factors facilitates reinitiation by remaining very stably bound to the promoter. This behavior contrasts markedly with the highly dynamic behavior of RNA Pol I transcription complexes in vivo, which undergo cycles of disassembly/reassembly at the promoter for each round of transcription. To determine whether the dynamic behavior of the Pol II machinery in vivo is fundamentally different from that of Pol I and whether the static behavior of Pol II factors in vitro fully recapitulates their behavior in vivo, we used fluorescence recovery after photobleaching (FRAP). Surprisingly, we found that all or nearly all of the TATA-binding protein (TBP) population is highly mobile in vivo, displaying FRAP recovery rates of <15 s. These high rates require the activity of the TBP-associated factor Mot1, suggesting that TBP/chromatin interactions are destabilized by active cellular processes. Furthermore, the distinguishable FRAP behavior of TBP and TBP-associated factor 1 indicates that there are populations of these molecules that are independent of one another. The distinct FRAP behavior of most Pol II factors that we tested suggests that transcription complexes assemble via stochastic multistep pathways. Our data indicate that active Pol II PICs can be much more dynamic than previously considered.

SUBMITTER: Sprouse RO

PROVIDER: S-EPMC2533185 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Regulation of TATA-binding protein dynamics in living yeast cells.

Sprouse Rebekka O RO Karpova Tatiana S TS Mueller Florian F Dasgupta Arindam A McNally James G JG Auble David T DT

Proceedings of the National Academy of Sciences of the United States of America 20080902 36

Although pathways for assembly of RNA polymerase (Pol) II transcription preinitiation complexes (PICs) have been well established in vitro, relatively little is known about the dynamic behavior of Pol II general transcription factors in vivo. In vitro, a subset of Pol II factors facilitates reinitiation by remaining very stably bound to the promoter. This behavior contrasts markedly with the highly dynamic behavior of RNA Pol I transcription complexes in vivo, which undergo cycles of disassembly ...[more]

PMID: 18765812

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Project description:Despite being one of the first eukaryotic transcriptional regulatory elements identified, the sequence of a native TATA box and its significance remain elusive. Applying criteria associated with TATA boxes we queried several Saccharomyces genomes and arrived at the consensus TATA(A/T)A(A/T)(A/G). Approximately 20% of yeast genes contain a TATA box. Strikingly, TATA box-containing genes are associated with responses to stress, are highly regulated, and preferentially utilize SAGA rather than TFIID when compared to TATA-less promoters. Transcriptional regulation in yeast appears to be mechanistically bipolar, possibly reflecting a need to balance inducible stress-related responses with constitutive housekeeping functions. A strain containing amino terminal HA-tagged TBP and its parental untagged counterpart BY4741 (Resgen) were grown at 23?C in CSM to OD600 = 0.8-1.0. Cultures were shifted to 37?C for 5 minutes (to mimic heat treatments used elsewhere (Huisinga and Pugh, 2004)), then crosslinked at 23?C with 1% formaldehyde. ChIP was performed as described previously (Strahl-Bolsinger et al., 1997) with the some modification. Following cell disruption with glass beads, the chromatin was partially purified by centrifugation (Kurdistani and Grunstein, 2003). HA-TBP was immunopurified with 12CA5 antibody. After elution and reversal of the crosslinks, samples were subjected to non-specific amplification, labeling, and array hybridization as described (Bohlander et al., 1992; Chitikila et al., 2002). The amplification procedure was modified as follows. Following 15 round B amplification cycles, DNA was purified via QIAquick PCR Purification kit. Ten cycles were used in round C. Intergenic microarrays were generated by PCR amplification of entire intergenic regions of all yeast genes, then spotted onto glass slides. Spot intensities were filtered to remove any signal that was less then one standard deviation above local background. Three replicates were performed and their log2 ratios (enriched/input) were averaged. Keywords = Chromatin Immunoprecipitation Keywords = genome-wide binding Keywords: other

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Regulation of TATA-binding protein dynamics in living yeast cells.

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Regulation of TATA-binding protein dynamics in living yeast cells.

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