Transcription Elongation (transcription + elongation)

Distribution by Scientific Domains


Selected Abstracts


Crystallization and preliminary crystallographic analysis of eukaryotic transcription and mRNA export factor Iws1 from Encephalitozoon cuniculi

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 2 2010
Michael Koch
Transcription elongation by eukaryotic RNA polymerase II requires the coupling of mRNA synthesis and mRNA processing and export. The essential protein Iws1 is at the interface of these processes through its interaction with histone chaperone and elongation factor Spt6 as well as with complexes involved in mRNA processing and export. Upon crystallization of the evolutionarily conserved domain of Iws1 from Encephalitozoon cuniculi, four different crystal forms were obtained. Three of the crystal forms belonged to space group P21 and one belonged to space group P2221. Preliminary X-ray crystallographic analysis of one of the crystal forms allowed the collection of data to 2.5,Å resolution. [source]


A simple in vivo assay for measuring the efficiency of gene length-dependent processes in yeast mRNA biogenesis

FEBS JOURNAL, Issue 4 2006
Macarena Morillo-Huesca
We have developed a simple reporter assay useful for detection and analysis of mutations and agents influencing mRNA biogenesis in a gene length-dependent manner. We have shown that two transcription units sharing the same promoter, terminator and open reading frame, but differing in the length of their 3,-untranslated regions, are differentially influenced by mutations affecting factors that play a role in transcription elongation or RNA processing all along the transcription units. In contrast, those mutations impairing the initial steps of transcription, but not affecting later steps of mRNA biogenesis, influence equally the expression of the reporters, independently of the length of their 3,-untranslated regions. The ratio between the product levels of the two transcription units is an optimal parameter with which to estimate the efficiency of gene length-dependent processes in mRNA biogenesis. The presence of a phosphatase-encoding open reading frame in the two transcription units makes it very easy to calculate this ratio in any mutant or physiological condition. Interestingly, using this assay, we have shown that mutations in components of the SAGA complex affect the level of mRNA in a transcript length-dependent fashion, suggesting a role for SAGA in transcription elongation. The use of this assay allows the identification and/or characterization of new mutants and drugs affecting transcription elongation and other related processes. [source]


Transcription elongation factor S-II maintains transcriptional fidelity and confers oxidative stress resistance

GENES TO CELLS, Issue 10 2003
Hiroshi Koyama
Background:, During transcription elongation, RNA polymerase II is arrested on the template when incorrect ribonucleotides are incorporated into the nascent transcripts. Transcription factor S-II enhances the excision of these mis-incorporated nucleotides by RNA polymerase II and stimulates transcription elongation in vitro. This mechanism is considered to be transcriptional proof-reading, but its physiological relevance remains unknown. Results:, We report that S-II contributes to the maintenance of transcriptional fidelity in vivo. We employed a genetic reporter assay utilizing a mutated lacZ gene from which active ,-galactosidase protein is expressed when mRNA proof-reading is compromised. In S-II-disrupted mutant yeasts, ,-galactosidase activity was ninefold higher than that in wild-type. The S-II mutant exhibited sensitivity to oxidants, which was suppressed by introduction of the S-II gene. The mutant S-II proteins, which are unable to stimulate transcription by RNA polymerase II in vitro, did not suppress the sensitivity of the mutants to oxidative stress or maintain transcriptional fidelity. Conclusion:, These results suggest that S-II confers oxidative stress resistance by providing an mRNA proof-reading mechanism during transcription elongation. [source]


Level of MYC overexpression in pediatric Burkitt's lymphoma is strongly dependent on genomic breakpoint location within the MYC locus

GENES, CHROMOSOMES AND CANCER, Issue 2 2004
Monika Wilda
Increased transcriptional activity of the MYC gene is a characteristic feature of Burkitt's lymphoma. Aberrant MYC expression is caused by (1) chromosomal translocation to one of the loci carrying an immunoglobulin gene, (2) mutation within the translocated allele, (3) loss of the block to transcription elongation, or (4) promoter shift. To investigate the influence of breakpoint locations within the MYC gene on MYC transcript levels, we determined both the precise genomic MYC/IGH breakpoints and the amount of MYC mRNA in 25 samples of pediatric Burkitt's lymphoma with translocation t(8;14)(q24;q32). Patients with breakpoints that were 5, from MYC exon 1 had significantly lower expression of MYC than did patients who had a breakpoint within exon 1 or intron 1 (P < 0.05 and 0.005, respectively). The highest mRNA level of MYC (1,006 copies per 100 copies ABL1) was detected in patients with loss of the first exon and transcription initiation from a cryptic P3 promoter within the first intron of the MYC gene. In contrast, there was no obvious correlation between breakpoint locations within the IgH locus and the amount of MYC mRNA. © 2004 Wiley-Liss, Inc. [source]


Distinct regions of cyclinT1 are required for binding to CDK9 and for recruitment to the HIV-1 Tat/TAR complex

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue S36 2001
Alessandro Fraldi
Abstract Tat-mediated activation of the HIV-1 promoter activity requires Tat-dependent recruitment of the cyclinT1/CDK9 complex (P-TEFb) to the transacting element (TAR) RNA. Tat interaction with the cyclinT1, the regulatory partner of CDK9, results in a specific recruitment of the heterodimer CycT1/CDK9 complex to TAR, whereby it promotes transcription elongation of the HIV-1 LTR-mediated transcription. Using the yeast two-hybrid protein interaction assay we analyzed the binding between cyclinT1 and CDK9. Moreover, using a modified three-hybrid yeast interaction system, we analyzed the recruitment of CycT1 to the Tat/TAR complex. The data presented here demonstrated that distinct domains of cyclinT1 interact with CDK9 and Tat/TAR in vivo. These findings will be instrumental for the designing of proper dominant-negative P-TEFb components capable to interfere with Tat function. J. Cell. Biochem. Suppl. 36: 247,253, 2001. © 2001 Wiley-Liss, Inc. [source]


Catalytic activity of Cdk9 is required for nuclear co-localization of the Cdk9/cyclin T1 (P-TEFb) complex

JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2003
Giuliana Napolitano
Cdk9 and its binding partner cyclin T1 comprise the positive elongation factor b (P-TEFb). P-TEFb phosphorylates the RNA polymerase II carboxyl-terminal-domain (CTD) allowing efficient transcription elongation. Recent studies showed that Cdk9 is a predominant nuclear protein, and here we investigated the functional requirement for nuclear localization of Cdk9. We found that the catalytic inactive kinase mutant (Cdk9dn) fails to accumulate in the nucleus showing a diffuse sub-cellular localization. In addition to the catalityc activity, nuclear localization of Cdk9 protein requires the presence of the phospho-acceptor sites at the C-terminus tail. Finally, enforced expression of wild-type cyclinT1, which enhances nuclear localization of Cdk9wt, fails to direct the Cdk9 mutants to the nucleus. Collectively, these findings implicate that nuclear localization of Cdk9 requires auto-phosphorylation of the kinase, and highlight the presence of a regulatory mechanism underlying the nuclear localization of the P-TEFb complex. J. Cell. Physiol. 197: 1,7, 2003© 2003 Wiley-Liss, Inc. [source]