The RNA polymerase genes rpoBC1C2 (the rpoB and rpoC2 are incomplete) of the cyanobacterium Nostoc commune have been cloned, sequenced, and expressed both in vivo and in vitro using E. coli systems. The rpo genes encode large subunits of DNA-dependent RNA polymerase. Two genes in N. commune, rpoC1 and rpoC2, correspond to rpoC of E. coli, which indicates a divergent evolution of RNA polymerase. The rpoBC1C2genes of Nostoc are linked in the order of rpoB, rpoC1, and rpoC2, and are transcribed differently from the corresponding rpo genes of E. coli. In E. coli the rpoBC genes are co-transcribed, together with two ribosomal protein genes. The Nostoc rpoB gene is transcribed by one promoter while the rpoC1C2 genes are co-transcribed by another promoter. Northern analysis of Nostoc RNA revealed two transcripts (3.1 and 5.6 kb), which were specific for the rpoB and rpoC1C2 genes, respectively. SDS-PAGE, Coomassie staining and immunoanalysis detected two polypeptides with molecular weights of 72 and 94 kd when the cloned rpoBC1C2 fragment was expressed in E. coli systems. These two polypeptides were assigned as products of rpoC1 and rpC2, respectively. The transcription of RNA polymerase genes of N. commune is sensitive to water stress (drying). The rpo transcription ceases upon drying and resumes after the dried cells have been rewetted for more than 5 min. The RNA polymerase enzyme itself, however, is stable under the same drying conditions. / Ph. D.

Indiana University-Purdue University Indianapolis (IUPUI) / RNA Polymerase II (RNAPII) is responsible for transcription of messenger RNA (mRNA) and many small non-coding RNAs. Progression through the RNAPII transcription cycle is orchestrated by combinatorial posttranslational modifications of the C-terminal domain (CTD) of the largest subunit of RNAPII, Rpb1, consisting of the repetitive sequence (Y1S2P3T4S5P6S7)n. Disruptions of proteins that control CTD phosphorylation, including the phosphatase Rtr1, cause defects in gene expression and transcription termination. There are two described RNAPII termination mechanisms. Most mRNAs are terminated by the polyadenylation-dependent cleavage and polyadenylation complex. Most short noncoding RNAs are terminated by the Nrd1 complex. Nrd1-dependent termination is coupled to RNA 3' end processing and/or degradation by Rrp6, a nuclear specific subunit of the exosome. The Rrp6-containing form a 3'-5' exonuclease complex that regulates diverse aspects of nuclear RNA biology including 3' end processing and degradation of a variety of noncoding RNAs (ncRNAs). It remains unclear whether Rrp6 is directly involved in termination. We discovered that deletion of RRP6 promotes extension of multiple Nrd1-dependent transcripts resulting from improperly processed 3' RNA ends and faulty transcript termination at specific target genes. Defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of Nrd1 depletion from the nucleus. Our data indicate Rrp6 acts with Nrd1 globally to promote transcription termination in addition to RNA processing and/or degradation. Furthermore, we found that deletion of the CTD phosphatase Rtr1 shortens the distance of transcription before Nrd1-dependent termination of specific regulatory antisense transcripts (ASTs), increases Nrd1 occupancy at these sites, and increases the interaction between Nrd1 and RNAPII. The RTR1/RRP6 double deletion phenocopies an RRP6 deletion, indicating that the regulation of ASTs by Rtr1 requires Rrp6 activity and the Nrd1 termination pathway.