No description available.

No description available.

No description available.

A family of E. coli - P. ruminis shuttle-plasmids was constructed to allow the isolation and characterization of gene promoters from the rumen bacterium P. ruminis. The promoter rescue plasmid pBK was used to isolate a total of 4 genomic DNA fragments that promoted transcription in P. ruminis strains 0/10. These promoters, and an additional promoter, previously isolated from P. ruminis strain OR38 (Schoep, 1999), were identified by their ability to initiate expression of a promoterless ermAM gene in P. ruminis. Within 4 of the fragments, a total of 5 transcription start sites were identified in P. ruminis using a novel, fluorescent-primer extension analysis protocol. Comparison of promoters isolated in this and previous studies revealed a strong consensus RNA polymerase DNA-binding motif, including the well characterized –35 and –10 elements. Consensus sequences established for these elements were: TTgacA and AtAATAta respectively, where bold upper-case font, regular upper-case, and lower-case fonts represent conservation in 100%, 80%, and 70% of promoters respectively. The −10 and −35 motifs were interspaced by 16 – 18 nt. Among the newly identified promoters, the consensus for the –10 element was extended one nucleotide upstream and downstream of the standard hexamer (boxed). These motifs were similar to those recognized by eubacterial RNA polymerase containing the σ70-like factor. Promoters also contained possible UP elements, and were significantly more curved than protein-coding regions. Additional plasmid vectors were constructed, to allow the use of both the quantitative SYBR green real time PCR and ß-glucuronidase assays, to examine 4 promoters in depth. This showed a wide range of promoter strengths within the group. However, no correlation was found between the composition and context of elements within P. ruminis promoters, and promoter strength. A mutation within the –35 element of one promoter revealed that promoter strength, and the choice of transcription start site were both sensitive to single nucleotide

Diss. (sammanfattning) Umeå : Univ., 2003. / Härtill 3 uppsatser.

<p>Eukaryotic RNA polymerase II (RNAPII) not only synthesizes mRNA, but also coordinates transcription-related processes through the post-translational modification of its unique C-terminal repeat domain (CTD). The CTD is an RNAPII specific extension of the enzyme's largest subunit and consists of multiple repeating heptads with the consensus sequence Y₁S₂P₃T₄S₅P₆S₇. In <italic>Saccharomyces cerevisiae (Sc)</italic>, RNAPII committed to productive elongation is phosphorylated at the S₂ positions of the CTD, primarily by CTDK-I (composed of the CDK-like Ctk1, the cyclin-like Ctk2, and Ctk3) the principal elongation-phase CTD kinase in <italic>Sc</italic>. Although responsible for the bulk of S₂ phosphorylation <italic>in vivo</italic>, Ctk1 coexists with the essential kinase Bur1 which also contributes to S₂ phosphorylation during elongation. In higher eukaryotes there appears to be only one CTD S₂ kinase: P-TEFb, which had been suggested to reconstitute the activity of both of the <italic>Sc</italic> S₂ CTD kinases. Based on comparative genomics, we hypothesized that the previously-unstudied <italic>Drosophila</italic> CDK12 (dCDK12) and little-studied human CDK12 and CDK13 (hCDK12 and hCDK13) proteins are CTD elongation-phase kinases, the metazoan orthologs of yeast Ctk1. Using fluorescence microscopy we show that the distribution of dCDK12 on formaldehyde-fixed polytene chromosomes is virtually identical to that of hyperphosphorylated RNAPII, but is distinct from that of P-TEFb. Chromatin immunoprecipitation experiments confirm that dCDK12 is present on the transcribed regions of active <italic>Drosophila</italic> genes in a pattern reminiscent of a S₂ CTD kinase. Appropriately, we show that dCDK12, hCDK12, and hCDK13 purified from nuclear extracts manifest CTD kinase activity <italic>in vitro</italic> and associate with CyclinK, implicating it as the cyclin subunit of the kinase. Most importantly we demonstrate that RNAi knockdown of dCDK12 in <italic>Drosophila</italic> cell culture and hCDK12 in human cell lines alters the phosphorylation state of the CTD. In an effort to further characterize the transcriptional roles of human CDK12/CyclinK we overexpress, purify to near homogeneity, and characterize, full-length hCDK12/CyclinK. Additionally, we also identify hCDK12 associated proteins via mass spectrometry, revealing interactions with multiple RNA processing factors, and attempt to engineer an analog sensitive CDK12 human cell line. Overall, these results demonstrate that CDK12 is a major elongation-associated CTD kinase, the ortholog of yCtk1. Our findings clarify the relationships between two yeast CDKs, Ctk1 and Bur1, and their metazoan homologues and draw attention to major metazoan CTD kinase activities that have gone unrecognized and unstudied until now. Furthermore, the results suggest that hCDK12 affects RNA processing events in two distinct ways: Indirectly through generating factor-binding phospho-epitopes on the CTD of elongating RNAPII and directly through binding to specific factors.</p> / Dissertation

No description available.

The archaeal RNA polymerase (RNAP) is similar to the eukaryotic RNAP-II in terms of subunit composition and overall protein structure. Despite its similarity, a new archaeal-specific Rpo13 subunit has been identified. Rpo13 occupies a position in the enzyme which, in RNAP-II, is filled by the eukaryotic-specific Rpb5 jaw domain. It has therefore been proposed to contact DNA, where the positively charged C-terminal tail might mediate protein-DNA interactions. Furthermore, analysis of archaeal genomes has identified a homologue of the eukaryotic RNAP-III-specific RPC34 subunit. RPC34 may associate with the single archaeal RNAP, modulating the specificity of the archaeal RNAP and re-directing it to a subset of genes such as non-coding genes, in analogy to the RNAP-III/RPC34 eukaryotic system involved in the transcription of 5S rRNA, tRNAs and other small RNAs. More importantly, the association of RPC34 with the single archaeal RNAP would define an archaeal enzyme which acts as a precursor of the eukaryotic RNAP-III. Electrophoretic mobility shift assay (EMSA) analysis of purified Rpo13 protein by recombinant means subsequently incubated with a double-stranded (ds)DNA sequence reveals the formation of protein-DNA complexes, where Rpo13’s binding to DNA is non-sequence specific but discriminatory to dsDNA, as no binding is observed in the presence of single-stranded (ss)DNA. Also, it is found that the ma jor determinant of DNA binding is the Rpo13’s positively charged C-terminal tail, since DNA binding is abolished with a Rpo13 mutant deficient in this tail. Furthermore, neither ma jor groove nor minor groove interacting compounds have a major impact on Rpo13’s binding to DNA, suggesting that Rpo13 may associate with the negatively charged DNA phosphate backbone. Moreover, in vitro transcription assays indicate that a transcription product is observed upon RNAP incubation with a bubble DNA oligo shown to make Rpo13 contacts in the RNAP-DNA crystal structure. In addition, while a GST-pulldown experiment suggests the existence of an interaction between the archaeal RNAP and RCP34 in vitro, co-immunoprecipitation assays argue against the existence of such interaction from an in vivo point of view. Finally, a chromatin immunoprecipitation (ChIP)-sequencing approach to analyse Rpo13’s genomic distribution versus the one of the bulk RNAP was undertaken. While the gel filtration elution profile analysis of Rpo13 in the S. acidocaldarius cell extract versus the one of recombinant Rpo13 suggests that there is a free Rpo13 pool in the cell extract, indicating that Rpo13 may be acting as a transiently-associated RNAP subunit displaying a factor-like function, the ChIP-sequencing approach reveals that Rpo13 is a bona fide RNAP subunit since it co-localises from a genomic point of view with the bulk RNAP.

The Spt4-Spt5 heterodimer complex is one of the key transcription elongation factors for RNA polymerase II, and thus helps to regulate gene expression with either positive or negative stimulation. Spt5 is a part of the NusG family of proteins, and is universally conserved across all three domains of life. This complex is also noted to be involved in many other cellular functions, including chromatin folding, DNA repair, and 5’ cap recruitment; both subunits also play roles in cellular activity when not bound together. However, there is still a great deal of insight to gain about this compound’s functions. This report delves into a variety of previous studies on this complex, summarizing known facts. It will describe how the Spt4-Spt5 complex is actually involved in facilitating transcription for nearly every type of RNA polymerase known so far, and that the secondary characteristics define each homologous structure. The variety of laboratory techniques utilized in these studies will also be noted, and the functionality of this versatile complex will be conveyed as known.

No description available.