RESUMO
Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF-I. In exploring the functional significance of the promoter-proximal terminator, we found that TTF-I associates with the p300/CBP-associated factor PCAF, suggesting that TTF-I may target histone acetyltransferase to the rDNA promoter. We demonstrate that PCAF acetylates TAF(I)68, the second largest subunit of the TATA box-binding protein (TBP)-containing factor TIF-IB/SL1, and acetylation enhances binding of TAF(I)68 to the rDNA promoter. Moreover, PCAF stimulates RNA polymerase I (Pol I) transcription in a reconstituted in vitro system. Consistent with acetylation of TIF-IB/SL1 being required for rDNA transcription, the NAD(+)-dependent histone deacetylase mSir2a deacetylates TAF(I)68 and represses Pol I transcription. The results demonstrate that acetylation of the basal Pol I transcription machinery has functional consequences and suggest that reversible acetylation of TIF-IB/SL1 may be an effective means to regulate rDNA transcription in response to external signals.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição , RNA Polimerase I/biossíntese , Proteínas de Saccharomyces cerevisiae , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Acetilação , Acetiltransferases/antagonistas & inibidores , Animais , DNA Ribossômico , Regulação Enzimológica da Expressão Gênica , Inativação Gênica , Histona Acetiltransferases , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Camundongos , Ligação Proteica , Subunidades Proteicas , RNA Polimerase I/genética , Proteínas Recombinantes/metabolismo , Sirtuína 2 , Sirtuínas , Proteína de Ligação a TATA-Box , Regiões Terminadoras Genéticas , Transativadores/metabolismo , Transcrição GênicaRESUMO
The separation of macromolecules in vertical rotors using linear or isokinetic sucrose gradients permits shorter centrifugation times and a higher sample capacity compared to swing-out rotors. In vertical rotors appropriate gradients give a resolution almost identical to that in swinging bucket rotors. This could be demonstrated for the isolation of polysomes and oligonucleosomes from rat liver. A microprocessor-controlled gradient former is presented which produces gradients of any desired shape. This device has been applied to prepare gradients with the desired linear or isokinetic shape after reorientation in the vertical tube during centrifugation.
Assuntos
Centrifugação com Gradiente de Concentração/instrumentação , Animais , Fígado/análise , Microcomputadores , Nucleossomos/análise , Polirribossomos/análise , Ratos , SacaroseRESUMO
This paper is a report about a rotation-viscometer with a submerged rotor which has been developed for measuring the viscosity of biological macromolecules. The device avoids the effects of surface disturbance. The rotor is centered and height-balanced electromagnetically and is controlled by a light barrier. The driving force is a rotating electromagnetic field and rotor revolution periods are measured by an electronic timer controlled by a second ligh barrier. Shearing effects are negligible if very slow revolutions are pre-selected; thus, intrinsic viscosity values for DNA can be obtained by merely extrapolating the concentration dependence. In contrast to DNA, chromatin has a very low viscosity with almost no dependence on concentration. If the ionic strength of a chromatin solution is decreased, the viscosity increases due to structural unfolding.
