Elongation rate of rDNA by RNAP I

Value 95 nucleotides/sec
Organism Mammalian tissue culture cell
Reference Dundr M, Hoffmann-Rohrer U, Hu Q, Grummt I, Rothblum LI, Phair RD, Misteli T. A kinetic framework for a mammalian RNA polymerase in vivo. Science. 2002 Nov 22 298(5598):1623-6. p.1625 left column bottom paragraphPubMed ID12446911
Method "In order to visualize RNA pol I in vivo, [researchers] tagged several RNA pol I components...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...In order to specifically visualize the fraction of RNA pol I subunits engaged in elongation and to obtain quantitative information about dynamics of elongation, [researchers] applied a modified bleaching method termed “iFRAP” (inverse FRAP). In iFRAP, the entire cell nucleus with the exception of a small region of interest containing a few sites of rDNA transcription is bleached (Fig. 3). In this way, a snapshot of the GFP fusion protein at sites of ribosomal gene expression at the time of bleaching is obtained. The loss of fluorescence is a direct indicator of the dissociation kinetics of a protein from ribosomal genes (Fig. 3)...To obtain quantitative information about assembly and elongation kinetics of RNA pol I components, researchers analyzed the iFRAP data with the use of a kinetic model based on standard principles of chemical kinetics (Fig. 4A) (14). The cycle of RNA pol I components can be described as a system of differential equations containing kinetic parameters of the pol I subunits as variables. For quantitation purposes, [they] assumed ~100 polymerases per active gene and a total number of active ribosomal genes of ~100 to 120 per nucleus (15–17). The known size of the elongating pool (~100 polymerases per gene) and a rough estimate of the absolute abundance of GFP tagged molecules in FCs [Within the mammalian nucleus, the tandemly repeated ribosomal genes are localized specifically in morphologically distinct nucleolar structures termed “fibrillar centers” (FCs)] were used as stringent constraints for obtaining the best fits to the model."
Comments "[Researchers] conclude from this analysis that the elongation phase of RNA pol I on ribosomal genes takes ~140 s, corresponding to an elongation rate of 95 nucleotides/s for a human rDNA gene of 13.3 kb [for length of gene see Gonzalez et al 1995, PUBMEDID 7557999]."
Entered by Uri M
ID 105113