TIF-IC, a factor involved in both transcription initiation and elongation of RNA polymerase I (original) (raw)
Related papers
Mammalian RNA polymerase I exists as a holoenzyme with associated basal transcription factors
Journal of Molecular Biology, 1998
Transcription initiation of ribosomal RNA genes requires RNA polymerase I (Pol I) and auxiliary factors which either bind directly to the rDNA promoter, e.g. TIF-IB/SL1 and UBF, or are assembled into productive transcription initiation complexes via interaction with Pol I, e.g. TIF-IA, and TIF-IC. Here we show that all components required for speci®c rDNA transcription initiation are capable of physical interaction with Pol I in the absence of DNA and can be co-immunoprecipitated with antibodies against de®ned subunits of murine Pol I. Sucrose gradient centrifugation and fractionation on gel ®ltration columns reveals that approximately 10% of cellular Pol I elutes as a de®ned complex with an apparent molecular mass of >2000 kDa. The large Pol I complex contains saturating levels of TIF-IA, TIF-IB and UBF, but limiting amounts of TIF-IC. In support of the existence of a functional complex between Pol I and basal factors, the large complex is transcriptionally active after complementation with TIF-IC. The results suggest that, analogous to class II gene transcription, a pre-assembled complex, the``Pol I holoenzyme'', exists that appears to be the initiation-competent form of Pol I.
Proceedings of the National Academy of Sciences, 2014
Transcription factors IIS (TFIIS) and IIF (TFIIF) are known to stimulate transcription elongation. Here, we use a single-molecule transcription elongation assay to study the effects of both factors. We find that these transcription factors enhance overall transcription elongation by reducing the lifetime of transcriptional pauses and that TFIIF also decreases the probability of pause entry. Furthermore, we observe that both factors enhance the processivity of RNA polymerase II through the nucleosomal barrier. The effects of TFIIS and TFIIF are quantitatively described using the linear Brownian ratchet kinetic model for transcription elongation and the backtracking model for transcriptional pauses, modified to account for the effects of the transcription factors. Our findings help elucidate the molecular mechanisms by which transcription factors modulate gene expression.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1987
The sites required for the formation of a stable transcription initiation complex and for the initiation of transcription of rat rDNA in vitro were examined. A series of 5' deletion mutants of the rat transcription initiation region (-167 through + 638) were constructed. These mutants were examined for their ability to support the faithful initiation of transcription in vitro. Mutants which contain less than 31 nucleotides upstream of the initiation site (+ 1) were unable to support detectable initiation of transcription. In this transcription system a series of deletion mutants from-167 to-31 were transcribed with equal efficiency when assayed individually. On the other hand, when the wild-type and mutant templates were compared in order-of-addition assays, they were found to he unequal. The incubation of an extract with a wild-type template, prior to the addition of nucleotides, precluded transcription of any second template added after the preincubation step. However, the preincubation of extract with mutants of the region upstream of the core promoter, from-122 to-31, did not preclude transcription of a wild-type template added after the preincubation step. Formation of the stable preinitiation complex was found to require the region between-122 and-167.
Molecular and Cellular Biology, 2002
In the small, free-living amoeba Acanthamoeba castellanii, rRNA transcription requires, in addition to RNA polymerase I, a single DNA-binding factor, transcription initiation factor IB (TIF-IB). TIF-IB is a multimeric protein that contains TATA-binding protein (TBP) and four TBP-associated factors that are specific for polymerase I transcription. TIF-IB is required for accurate and promoter-specific initiation of rRNA transcription, recruiting and positioning the polymerase on the start site by protein-protein interaction. In A. castellanii, partially purified TIF-IB can form a persistent complex with the ribosomal DNA (rDNA) promoter while homogeneous TIF-IB cannot. An additional factor, TIF-IE, is required along with homogeneous TIF-IB for the formation of a stable complex on the rDNA core promoter. We show that TIF-IE by itself, however, does not bind to the rDNA promoter and thus differs in its mechanism from the upstream binding factor and upstream activating factor, which carry out similar complex-stabilizing functions in vertebrates and yeast, respectively. In addition to its presence in impure TIF-IB, TIF-IE is found in highly purified fractions of polymerase I, with which it associates. Renaturation of polypeptides excised from sodium dodecyl sulfate-polyacrylamide gels showed that a 141-kDa polypeptide possesses all the known activities of TIF-IE.
Nucleic Acids Research, 1993
TIF-IB is a transcription factor which interacts with the mouse ribosomal gene promoter and nucleates the formation of an initiation complex containing RNA polymerase I (Pol 1). We have purified this factor to near homogeneity and demonstrate that TIF-IB is a large complex (<200 kDa) which contains several polypeptides. One of the subunits present in this protein complex is the TATA-binding protein (TBP) as revealed by copurification of TIF-IB activity and TBP over different chromatographic steps including immunoaffinity purification. In addition to TBP, three tightly associated proteins (TAFs-l) with apparent molecular weights of 95, 68, and 48 kDa are contained in this multimeric complex. This subunit composition is similar-but not identical-to the analogous human factor SL1. Depletion of TBP from TIF-IB-containing fractions by immunoprecipitation eliminates TIF-IB activity. Neither TBP alone nor fractions containing other TBP complexes are capable of substituting for TIF-IB activity. Therefore, TIF-IB is a unique complex with Pol I-specific TAFs distinct from other TBPcontaining complexes. The identification of TBP as an integral part of the murine rDNA promoter-specific transcription initiation factor extends the previously noted similarity of transcriptional initiation by the three nuclear RNA polymerases and underscores the importance of TAFs in determining promoter specificity.
Molecular and cellular biology, 1993
Alterations in the rate of cell proliferation are accompanied by changes in the transcription of rRNA genes. In mammals, this growth-dependent regulation of transcription of genes coding for rRNA (rDNA) is due to reduction of the amount or activity of an essential transcription factor, called TIF-IA. Extracts prepared from quiescent cells lack this factor activity and, therefore, are transcriptionally inactive. We have purified TIF-IA from exponentially growing cells and have shown that it is a polypeptide with a molecular mass of 75 kDa which exists as a monomer in solution. Using a reconstituted transcription system consisting of purified transcription factors, we demonstrate that TIF-IA is a bona fide transcription initiation factor which interacts with RNA polymerase I. Preinitiation complexes can be assembled in the absence of TIF-IA, but formation of the first phosphodiester bonds of nascent rRNA is precluded. After initiation, TIF-IA is liberated from the initiation complex a...
Cell cycle and growth stimuli regulate different steps of RNA polymerase I transcription
Gene, 2016
Transcription of the ribosomal RNA genes (rDNA) by RNA polymerase I (Pol I) is a major control step for ribosome synthesis and is tightly linked to cellular growth. However, the question of whether this process is modulated primarily at the level of transcription initiation or elongation is controversial. Studies in markedly different cell types have identified either initiation or elongation as the major control point. In this study, we have re-examined this question in NIH3T3 fibroblasts using a combination of metabolic labeling of the 47S rRNA, chromatin immunoprecipitation analysis of Pol I and overexpression of the transcription initiation factor Rrn3. Acute manipulation of growth factor levels altered rRNA synthesis rates over 8-fold without changing Pol I loading onto the rDNA. In fact, robust changes in Pol I loading were only observed under conditions where inhibition of rDNA transcription was associated with chronic serum starvation or cell cycle arrest. Overexpression of ...