Content area
Full Text
We have analyzed the kinetics of assembly and elongation of the mammalian RNA polymerase I complex on endogenous ribosomal genes in the nuclei of living cells with the use of in vivo microscopy. We show that components of the RNA polymerase I machinery are brought to ribosomal genes as distinct subunits and that assembly occurs via metastable intermediates. With the use of computational modeling of imaging data, we have determined the in vivo elongation time of the polymerase, and measurements of recruitment and incorporation frequencies show that incorporation of components into the assembling polymerase is inefficient. Our data provide a kinetic and mechanistic framework for the function of a mammalian RNA polymerase in living cells.
Within the mammalian nucleus, the tandemly repeated ribosomal genes are localized specifically in morphologically distinct nucleolar structures termed "fibrillar centers" (FCs), where they are transcribed exclusively by RNA polymerase I (1-3). FCs are naturally occurring gene arrays enriched in components of the RNA pol I machinery and are therefore an ideal system to visualize and to quantitatively study the dynamics of an RNA polymerase on its endogenous target in living cells.
In order to visualize RNA pol I in vivo, we tagged several RNA pol I components, including preinitiation factors Upstream Binding Factor 1 (UBF1), UBF2, and Transcription Associated Factor,48 (TAF,48), assembly factors Polymerase Associated Factor 53 (PAF53) and Transcription Initiation Factor-IA (TIF-IA/Rrn3), and the subunits of the polymerase (RPA194, RPA43, RPA40, and RPA 16) with the green fluorescent protein (GFP). The fusion proteins were expressed transiently or stably in CMT3 monkey kidney cells where fusion proteins accumulated in the nucleolus in multiple foci indicative of FCs (Fig. 1, A and B). The punctate sites of accumulation were confirmed to be endogenous ribosomal genes by fluorescence in situ hybridization using a specific probe against the nascent 5' External Transcribed Spacer (ETS) core segment of preribosomal RNA (pre-rRNA) (Fig. 1A) (4). Intact ribosomal DNA (rDNA) transcription in cells expressing tagged RNA pol I components was confirmed by incorporation of 5-bromouridine 5'-triphosphate (BrUTP) in in situ runon assays (Fig. 1B). As observed for endogenous RNA pol I components by antibody staining, a weak diffuse nucleoplasmic signal and a cytoplasmic pool was detected for all fusion proteins (Fig. 1, A and B)...