RESUMEN
RNA polymerase III (Pol III) occurs in two versions, one containing the POLR3G subunit and the other the closely related POLR3GL subunit. It is not clear whether these two Pol III forms have the same function, in particular whether they recognize the same target genes. We show that the POLR3G and POLR3GL genes arose from a DNA-based gene duplication, probably in a common ancestor of vertebrates. POLR3G- as well as POLR3GL-containing Pol III are present in cultured cell lines and in normal mouse liver, although the relative amounts of the two forms vary, with the POLR3G-containing Pol III relatively more abundant in dividing cells. Genome-wide chromatin immunoprecipitations followed by high-throughput sequencing (ChIP-seq) reveal that both forms of Pol III occupy the same target genes, in very constant proportions within one cell line, suggesting that the two forms of Pol III have a similar function with regard to specificity for target genes. In contrast, the POLR3G promoter--not the POLR3GL promoter--binds the transcription factor MYC, as do all other promoters of genes encoding Pol III subunits. Thus, the POLR3G/POLR3GL duplication did not lead to neo-functionalization of the gene product (at least with regard to target gene specificity) but rather to neo-functionalization of the transcription units, which acquired different mechanisms of regulation, thus likely affording greater regulation potential to the cell.
Asunto(s)
Duplicación de Gen , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Cromatina/metabolismo , Evolución Molecular , Genoma , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Filogenia , Regiones Promotoras Genéticas , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Polimerasa III/química , Alineación de Secuencia , Vertebrados/genéticaAsunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/biosíntesis , ARN Mensajero/biosíntesis , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Animales , Biopolímeros/metabolismo , ADN Polimerasa II/metabolismo , Humanos , Unión Proteica/fisiología , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Rho-dependent transcription termination at the phage lambda tR1 terminator is governed primarily by the upstream rut element that encodes two RNA regions rutA and rutB. The two regions are separated by the boxB RNA motif, which is believed to be dispensable for Rho activity but serves as a binding site for lambda N protein in the antitermination process. By using a minimal in vivo termination system, we show that the intervening boxB RNA motif has a double function in the mechanisms of termination/antitermination at lambdatR1. As a folded hairpin structure, it acts as a clamp that holds rutA and rutB side by side for optimal interactions with Rho leading to efficient termination. Conversely, the binding of N protein to boxB induces antitermination at lambdatR1 by preventing access of Rho to the rut sequences. This dual role was clearly shown in vivo by studying the effects of multiple mutations within the boxB hairpin stem on transcription termination and by substituting the N/boxB couple with the unrelated coat protein of phage MS2 and its stem-loop RNA binding site.
