RESUMO
The ubiquitous transcription factor upstream stimulatory factor (USF) 1 is a member of the bzHLH (leucine zipper-basic-helix-loop-helix) family, which is structurally related to the Myc family of proteins. It plays a role in the regulation of many genes, including the cyclin B1 gene, which is active during the G2/M and M phases of the cell cycle and may also play a role in the regulation of cellular proliferation. We show that the affinity of recombinant USF-1 for DNA is greatly increased by treatment with active cyclin A2-p34(cdc2) or cyclin B1-p34(cdc2) complexes and that its interaction with DNA is dependent on p34(cdc2)-mediated phosphorylation. We have localized the phosphorylation site(s) to a region that lies outside the minimal DNA-binding domain but overlaps with the previously identified USF-specific region. Deletion studies of USF-1 suggest that amino acids 143-197 regulate DNA-binding activity in a phosphorylation-dependent manner.
Assuntos
Ciclinas/metabolismo , Proteínas de Ligação a DNA , DNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Proteína Quinase CDC2/metabolismo , Ciclo Celular , Ciclinas/genética , Sondas de DNA/genética , Feminino , Células HeLa , Humanos , Técnicas In Vitro , Mitose , Oócitos/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genética , Fatores Estimuladores Upstream , XenopusRESUMO
The stages and kinetics of transcription complex formation in a Saccharomyces cerevisiae in vitro transcription system were analysed using the anionic detergent sarkosyl. In contrast to findings from other systems, we were not able to distinguish between a fully formed pre-initiation complex and a 'rapid start' complex to which nucleotides were added. A further increase in resistance of transcription to sarkosyl was only observed 12 min after transcription initiation, by which time elongation was underway. Low concentrations of ATP, dATP or, surprisingly, the non-hydrolysable analogue ATPgammaS selectively stimulated transcription when present during assembly of transcription complexes, although the level of stimulation dropped when ATP was added progressively later. The effect of ATP did not correlate with the kinetics of template commitment, signifying that it functioned at a later stage than this, but prior to the full assembly of sarkosyl-resistant pre-initiation complexes. ATP also altered the sarkosyl resistance of initiating transcription complexes possibly by affecting a rate-limiting step leading to earlier appearance of elongated transcripts. This effect was antagonised by ATPgammaS, thus providing evidence that the stimulatory effect of ATP on pre-initiation complex formation and its effect on the lag between initiation and elongation phases are distinct.
Assuntos
Trifosfato de Adenosina/farmacologia , Saccharomyces cerevisiae/genética , Transcrição Gênica/efeitos dos fármacos , Cinética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , Sarcosina/análogos & derivados , Sarcosina/farmacologiaRESUMO
We have identified and partially characterized a novel class III transcription factor fraction (TFIIIE) from yeast nuclear extracts. TFIIIE is functionally distinct from the standard yeast transcription factor fractions, TFIIIB and TFIIIC. It is also different from either of the TFIIIB subfractions, B' and B". TFIIIE is essential for specific transcription of both tRNA and 5 S RNA genes, its activity is sensitive to proteinase K, and it exhibits an apparent sedimentation coefficient of 4.0 S when analyzed on glycerol gradients. In the case of a tRNA gene, TFIIIE does not play a role in the formation of stable preinitiation complexes containing TFIIIB and TFIIIC. It is required for single as well as multiple rounds of transcription, however. Thus, TFIIIE is involved in the utilization of stable transcription complexes, but its action is not restricted to reinitiation events.