Growth and differentiation of the neurites depends on long-distance transport of a specific set of mRNAs to restricted area and their local translation. Here we found that a TAR DNA-binding protein of

RNA polymerase II purified from the fission yeast Schizosaccharomyces pombe consists of 10 species of subunit polypeptide. We introduced a histidine cluster tag sequence into the chromosomal rpb1 and rpb3 genes, which encode subunit 1 (Rpb1) and subunit 3 (Rpb3), respectively, and purified the RNA polymerase by Ni 2؉ affinity chromatography. After stepwise dissociation of the Rpb1-and Rpb3-tagged RNA polymerases fixed on Ni 2؉ -resin by increasing concentrations of urea or guanidium hydrochloride, Rpb2-Rpb3-Rpb11 or Rpb2-Rpb3-Rpb11-Rpb10 complexes were obtained. Since the complex consisting of Rpb2, Rpb3, and Rpb11 cannot be dissociated even after treatment with 6 M urea buffer, we propose that this complex represents a core subassembly of the RNA polymerase II, analogous to the ␣ 2 ␤ complex in the assembly of Escherichia coli RNA polymerase. Both the Rpb2-Rpb3-Rpb11 complex and the free Rpb1 protein showed DNA binding activity, although the affinity was weaker compared with the intact RNA polymerase.Eukaryotic nuclear RNA polymerases are all multisubunit enzymes consisting of some 10 species of polypeptide. Among three types of nuclear RNA polymerase, RNA polymerase II (pol II) 1 is involved in the synthesis of mRNA and thus plays a central role in gene transcription. The genes coding for all the putative subunits have been cloned and sequenced for three organisms, human, the budding yeast Saccharomyces cerevisiae, and the fission yeast Schizosaccharomyces pombe; but for all these enzymes, little is known about the function(s) of each polypeptide.DNA blotting, ultraviolet cross-linking, and antibody inhibition studies indicated the involvement of subunit 1, the homologue of the E. coli RNA polymerase ␤Ј subunit, in DNA binding (1-5), whereas subunit 2, the homologue of the E. coli ␤ subunit, has binding sites for nucleotide substrates (6 -9) and DNA (1, 10, 11). S. cerevisiae pol II subunit 3 (RPB3) and the corresponding subunits from other organisms have a limited sequence similarity to the E. coli ␣ subunit. As in the case of the E. coli ␣ subunit, one enzyme protomer probably contains two molecules of RPB3, and this subunit forms a complex with RPB2 in an early step of the enzyme assembly in vivo (12, 13).The ␣-like sequence also exists in S. cerevisiae RPB11 and the corresponding subunits in other eukaryotes (14 -16). The RPB3 and RPB11 homologues of Arabidopsis thaliana pol II associate in vitro with each other (15). Molecular interaction between S. cerevisiae AC40 and AC19, which are RPB3 and RPB11 homologues present in both RNA polymerases I and III, was also suggested by genetic analysis (17).pol II purified from S. pombe contains 10 species of subunit (18), named Rpb1 to Rpb12 after the S. cerevisiae subunit nomenclature (19), lacking two subunits corresponding to RPB4 and RPB9 of S. cerevisiae pol II. The S. pombe genes coding for all 10 subunits have been cloned and sequenced (16, 20 -25). In the case of S. cerevisiae pol II, RPB4 and RPB7 are readily dissociated from the enzyme and form a heterodi...

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The essential Saccharomyces cerevisiae gene BDP1 encodes a subunit of RNA polymerase III (Pol III) transcription factor (TFIIIB); TATA box binding protein (TBP) and Brf1 are the other subunits of this three-protein complex. Deletion analysis defined three segments of Bdp1 that are essential for viability. A central segment, comprising amino acids 327 to 353, was found to be dispensable, and cells making Bdp1 that was split within this segment, at amino acid 352, are viable. Suppression of bdp1 conditional viability by overexpressing SPT15 and BRF1 identified functional interactions of specific Bdp1 segments with TBP and Brf1, respectively. A Bdp1 deletion near essential segment I was synthetically lethal with overexpression of PCF1-1, a dominant gain-of-function mutation in the second tetracopeptide repeat motif (out of 11) of the Tfc4 ( 131 ) subunit of TFIIIC. The analysis also identifies a connection between Bdp1 and posttranscriptional processing of Pol III transcripts. Yeast genomic library screening identified RPR1 as the specific overexpression suppressor of very slow growth at 37°C due to deletion of Bdp1 amino acids 253 to 269. RPR1 RNA, a Pol III transcript, is the RNA subunit of RNase P, which trims pre-tRNA transcript 5 ends. Maturation of tRNA was found to be aberrant in bdp1-⌬253-269 cells, and RPR1 transcription with the highly resolved Pol III transcription system in vitro was also diminished when recombinant Bdp1⌬253-269 replaced wild-type Bdp1. Physical interaction of RNase P with Bdp1 was demonstrated by coimmunoprecipitation and pull-down assays.RNA polymerase III (Pol III) transcribes genes encoding tRNAs, 5S rRNA, U6 snRNA, and other small RNAs. Accurate initiation of this transcription requires basal transcription factor IIIA (TFIIIA), TFIIIB, and TFIIIC. In the yeast Saccharomyces cerevisiae, TFIIIB is required for all Pol III transcription in vitro and in vivo, TFIIIC is required for all Pol III transcription in vivo, and TFIIIA is required only for 5S rRNA gene transcription. TFIIIB is composed of three subunits, TATA-binding protein (TBP), Brf1 (the TFIIB-related factor), and Bdp1; all three are also essential for Pol III transcription in vitro (7,12,14,21,27,28,33,40,51,52,65). In vivo analysis defines multiple interactions of TFIIIB with the rest of the S. cerevisiae Pol III transcription machinery: Brf1 and Bdp1 interact with Tfc4 (the second largest subunit of TFIIIC), and Brf1 also interacts with the RPC34 and RPC17 subunits of Pol III (3,5,19,20,39,47,52,64).In human cells, two TFIIIB-related assemblies have been identified (46,60). TFIIIB␣, which contains TBP, Bdp1 (previously called hTFIIIB150), and Brf2 (a hBrf1 paralogue previously called hTFIIIB50), is required for transcription of Pol III genes with upstream promoter elements, such as 7SK and U6 (53, 61). TFIIIB␤, containing TBP, Bdp1, and Brf1, is required for transcription of genes with internal promoters (53). Alternatively spliced variants of hBrf1 have also been noted (44). Human TFIIIB interacts with a subcomplex of P...