| 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 |