New approaches to structural studies of E. coli 5S RNA and its complexes with ribosomal proteins by NMR

Thesis/Dissertation · Fri Jan 01 04:00:00 EST 1988

OSTI ID:5527029

Gewirth, D T

The secondary structure of E. coli 5S RNA and its ribonucleoprotein complexes with L25 and L18 has been studied using nuclear magnetic resonance. Since purely spectroscopic techniques applied to either the intact 5S RNA or its nuclease-resistant fragment 1 (bases 1-11 and 69-120) had ceased to yield new information, new approaches to these structural studies were developed and applied. These are (1) the construction of fragment 1 molecules partially labeled with {sup 15}N, (2) site directed point and deletion mutagenesis to produce altered RNAs in vivo, and (3) transcription of full-length and deletion mutants of 5S in vitro using a T7 RNA Polymerase system. Partial {sup 15}N labeling patterns in fragment 1 were created by reconstituting fragment 1 from its labeled and unlabeled constituent oligonucleotides. This has allowed the firm assignment of 3 resonances in the terminal helix region and 2 resonances in the helix V region of fragment 1. Site-directed mutagenesis applied to the 5S RNA gene results in RNA molecules in which the spectroscopic effects of mutation are limited. The differences between normal and mutant spectra are used to make or confirm the assignments of resonances. A deletion mutant of 5S RNA called the pDG07 fragment, which lacks bases 11-69, was constructed and expressed in vivo. Its spectrum is similar to that of fragment 1 whose sequence it closely resembles. A point mutation at position 101 in this molecule disrupts the secondary structure of helix V and reduces the RNA's L25 binding activity. Complexes between L25 and the pDG07 fragment have allowed the first complete assignment scheme to be proposed for this molecule, including the assignment of all of the helix V resonances. 5S RNA molecules lacking bases 36-47 at the end of helix III show that the structure of helix III is altered and L18 binding is reduced.

• Research Organization: Yale Univ., New Haven, CT (USA)
• OSTI ID: 5527029
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
CONFIGURATION
ENZYMES
ESCHERICHIA COLI
HELICAL CONFIGURATION
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
MOLECULAR STRUCTURE
MUTATIONS
NITROGEN 15
NITROGEN ISOTOPES
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ODD-EVEN NUCLEI
OLIGONUCLEOTIDES
ORGANIC COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RESONANCE
RNA
RNA POLYMERASES
SPECTRA
STABLE ISOTOPES
TRACER TECHNIQUES
TRANSCRIPTION
TRANSFERASES