Effect of single base changes and the absence of modified bases in 16S RNA on the reconstitution and function of Escherichia coli 30S ribosomes
The gene coding for E. coli 16S rRNA was placed in pUC19 under the control of the strong class III T7 promoter, phi 10, by ligation of the 1490 bp BclI/BstEII fragment of the rrnB operon with appropriate synthetic oligodeoxynucleotides. Such constructs allowed efficient in vitro synthesis of full-length transcripts (up to 900 mol RNA/mol template) free of modified bases. The synthetic RNA could be assembled into 30S subunits upon addition of E. coli 30S ribosomal proteins. The particles co-sedimented with authentic 30S particles and were electron microscopically indistinguishable from them. Upon addition of 50S subunits, codon-dependent P-site binding of tRNA and codon-dependent polypeptide synthesis were >80% of 30S reconstituted from natural 16S RNA and >50% of isolated 30S. UV-induced crosslinking of P-site bound AcVal-tRNA to residue C1400 was preserved. Changing C1400 to A had little effect on reconstitution, P-site binding, or polypeptide synthesis. However, the substitution of C1499 by G markedly inhibited assembly. The effect on P-site binding and polypeptide synthesis is under study. These results show (1) none of the modified bases of 16S RNA are essential for protein synthesis, (2) substitution of A for C1400 has little functional effect, and (3) position 1400 may be important for ribosome assembly.
- Research Organization:
- Roche Research Center, Nutley, NJ
- OSTI ID:
- 6151282
- Report Number(s):
- CONF-870644-; TRN: 87-029039
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
POLYPEPTIDES
BIOSYNTHESIS
RIBOSOMES
BIOLOGICAL FUNCTIONS
TRANSFER RNA
CROSS-LINKING
ESCHERICHIA COLI
GENE OPERONS
GENETIC RADIATION EFFECTS
RIBOSOMAL RNA
ULTRAVIOLET RADIATION
BACTERIA
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
CELL CONSTITUENTS
CHEMICAL REACTIONS
ELECTROMAGNETIC RADIATION
FUNCTIONS
GENETIC EFFECTS
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANOIDS
PEPTIDES
POLYMERIZATION
PROTEINS
RADIATION EFFECTS
RADIATIONS
RNA
SYNTHESIS
560130* - Radiation Effects on Microorganisms