Analysis of functional sites in rRNA by RNA:DNA hybridization

Conference · Fri May 01 04:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6028951

Smith, J E; Cooperman, B S

Affinity labeling studies of functional sites in large RNAs require a method which allows (1) localization of the site, and (2) quantitation of the labeling at the site. They have developed such a method and evaluated it using the naturally methylated sites in E. coli rRNA as a model system. A (/sup 3/H-CH/sub 3/)-labeled site is localized by hybridizing labeled RNA to restriction fragments of complementary DNA. The RNA:DNA hybrids are separated by electrophoresis. The hybrid which contains the label then defines a region containing the site. Using several different restriction enzymes, they can localize a site to within 100 bases, or less. Precise quantitation of the labeling at a site is accomplished through inclusion of (/sup 14/C-uniformly-labeled RNA in the hybridization mixture. The second label allows correction of the amount of site-specific label for variation in yield of different hybrids. Application of this double-labeling approach yields quantitation of methylation that falls within about 10% of predicted values.

• Research Organization: Univ. of Pennsylvania, Philadelphia
• OSTI ID: 6028951
• Report Number(s): CONF-870644-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 46:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
CARBON 14 COMPOUNDS
CHEMICAL REACTIONS
DNA
DOUBLE LABELLING
ELECTROPHORESIS
ESCHERICHIA COLI
HYBRIDIZATION
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LABELLING
METHYLATION
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
RNA
TRACER TECHNIQUES
TRITIUM COMPOUNDS