Circularly permuted tRNAs as specific photoaffinity probes of ribonuclease P RNA structure

Journal Article · Fri Aug 06 04:00:00 EDT 1993 · Science (Washington, D.C.); (United States)

DOI:https://doi.org/10.1126/science.7688143· OSTI ID:5349262

Nolan, J M; Pace, N R ^[1]; Burke, D H ^[2]

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1. Indiana Univ., Bloomington, IN (United States)
2. Univ. of Colorado, Boulder, CO (United States)

Regions of Escherichia coli ribonuclease P (RNase P) RNA in proximity to a bound transfer RNA (tRNA) substrate were mapped by photoaffinity. A photoaffinity cross-linking reagent was introduced at specific sites in the interior of the native tRNA structure by modification of the 5[prime] ends of circularly permuted tRNAs (cptRNAs). The polymerase chain reaction was used for the production of cptRNA templates. After the amplification of a segment of a tandemly duplicated tRNA gene, the cptRNA gene was transcribed in vitro to produce cptRNA. Modified cptRNAs were cross-linked to RNase P RNA, and the conjugation sites in RNase P RNA were determined by primer extension. These sites occur in phylogenetically conserved structures and sequences and identify regions of the ribozyme that form part of the tRNA binding site. The use of circularly permuted molecules to position specific modifications is applicable to the study of many inter- and intramolecular interactions.

• OSTI ID: 5349262
• Journal Information: Science (Washington, D.C.); (United States), Journal Name: Science (Washington, D.C.); (United States) Vol. 261:5122; ISSN SCIEAS; ISSN 0036-8075
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
CELL CONSTITUENTS
CIRCULAR CONFIGURATION
CONFIGURATION
ENZYMES
ESTERASES
HYDROLASES
MOLECULAR STRUCTURE
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
PROTEINS
RIBOSOMAL RNA
RIBOSOMES
RNA
RNA-ASE
TRANSFER RNA