RNA folding during transcription by Escherichia coli RNA polymerase analyzed by RNA self-cleavage

Journal Article · Tue Aug 28 00:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00486a015· OSTI ID:5895008

Monforte, J A; Kahn, J D; Hearst, J E ^[1]

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1. Univ. of California, Berkeley (USA) Lawrence Berkeley Lab., CA (USA)

The authors have used a self-cleaving RNA molecule related to a subsequence of plant viroids (a hammerhead) to study the length-dependent folding of RNA produced during transcription by Escherichia coli RNA polymerase. Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues (3{prime}-deoxyNTP's or 3{prime}-O-methylNTP's). When the transcript can form the hammerhead structure it self-cleaves to give a truncated product. The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to those generated by using a noncleaving transcript or by using ({alpha}-thio)ATP in place of ATP. They have shown that 15-18 nucleotides (nt) of RNA past the cleavage point must be synthesized before the transcript can self-cleave within a ternary complex, whereas RNA freed from the complex by heating can cleave with only 3 or more nt present beyond the cleavage point. There are sequence-dependent as well as length-dependent effects. The results suggest that 12 {plus minus} 1 nt are sequestered within the ternary complex and are consistent with the presence of a DNA-RNA hybrid within the transcription bubble, as proposed by others. The results indicate that the hammerhead structure does not disrupt the hybrid. Self-cleaving of the transcript offers a simple structural probe for studying less well-characterized transcription complexes. The relevance of the results to models for transcription termination is discussed.

• DOE Contract Number: AC03-76SF00098
• OSTI ID: 5895008
• Journal Information: Biochemistry; (USA), Vol. 29:34; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
RNA
MOLECULAR STRUCTURE
RNA POLYMERASES
BIOCHEMICAL REACTION KINETICS
TRANSCRIPTION
MOLECULAR BIOLOGY
CONFORMATIONAL CHANGES
ESCHERICHIA COLI
GENE REGULATION
PHOSPHORUS 32
TEMPERATURE DEPENDENCE
VIRUSES
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
DAYS LIVING RADIOISOTOPES
ENZYMES
ISOTOPES
KINETICS
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PARASITES
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RADIOISOTOPES
REACTION KINETICS
TRANSFERASES
550201* - Biochemistry- Tracer Techniques