The Mode Of Action Of Cordycepin In The Inhibition Of Bacillus Subtilis Growth.

PhDBiochemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/185956/2/6400882.pd

Nucleotide Methylation Patterns in Eukaryotic mRNA

This chapter reviews that the use of enzymes for selective hydrolysis, coupled with high-resolution liquid chromatography for assay of products, provides an efficient means of determining the specific patterns of methylation in eukaryotic mRNA molecules. Continuous labeling with levels of L-[methyl-3H] methionine permits normal growth of Novikoff cells to examine the methylation of specific sites of cytoplasmic mRNA as a function of time. The main site of cytoplasmic mRNA labeling is at the second position (N'') of the 5'-terminal sequence. Data obtained by comparing methylnucleoside composition of these sequences and the ratio of doubly to singly O-methylated termini as a function of labeling time is consistent with a model in which m7G, N'm and the m6A located in the mRNA molecule are all products of nuclear methylation events. Subsequently there is a cytoplasmic methylation of some singly O-methylated structures at the second (N'') position yielding the doubly O-methylated structure. The kinetics of methyl labeling and the changing composition within the caps show a distinct pattern, possibly reflecting a selection or enrichment of a stable class of mRNA molecules, many of which contain the doubly labeled structure at their 5'-terminus and are of smaller size

The sensitivity of RNA polymerases I and II from Novikoff hepatoma (N1S1) cells to 3′-deoxyadenosine 5′-triphosphate

The synthesis of ribosomal precursor RNA in Novikoff hepatoma (NISI) cells is very sensitive to cordycepin (3′-dA). The synthesis of hnRNA, however, is resistant to inhibition by concentrations of 3′-dA that completely block the synthesis of 45S ribosomal RNA precursor. We have examined the RNA polymerases present in these cultured cells with regard to their sensitivity to cordycepin 5′-triphosphate (3′-dATP) in an effort to explain the differential inhibition of RNA synthesis observed in vivo. RNA polymerases I and II were characterized on the basis of their chromatographic behavior on DEAE-Sephadex, as well as the response of their enzymatic activities to ionic strength, the divalent metal ions Mn2$and Mg2$, and the toxin α-amanitin. For both enzymes the inhibition of in vitro RNA synthesis by 3′- dATP was competitive for ATP. The Km values for ATP and the Ki values for 3′-dATP for the two enzymes were quite similar. RNA polymerase II, the enzyme presumed responsible for hnRNA synthesis, was actually slightly more sensitive to 3′-dATP than RNA polymerase I, the enzyme presumed responsible for ribosomal precursor RNA synthesis. Similar data were obtained when the RNA polymerases were assayed in isolated nuclei. These results indicate that the differential inhibition of RNA synthesis caused by 3′-dA in vivo cannot be simply explained by differential sensitivity of RNA polymerases I and II to 3′-dATP