In vitro assembly of 30S and 70S bacterial ribosomes from 16S RNA containing single base substitutions, insertions, and deletions around the decoding site (C1400)
- 7 February 1989
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 28 (3) , 1002-1011
- https://doi.org/10.1021/bi00429a013
Abstract
An in vitro system developed for the site-specific mutagenesis of 16S RNA of Escherichia coli ribosomes [Krzyzosiak et al. (1987) Biochemistry 26, 2353-2364] was used to make 10 single base changes around C1400, a residue known to be at the decoding site. C1400 was replaced by U, A, or G, five single base deletions at and to either side of C1400 were made, and C or U was inserted next to C1400. Another mutant possessed seven additional nucleotides at the 3'' end of the 16S RNA such that a stem and loop involving the anti-Shine-Dalgarno sequence could form. Each of the mutnat RNAs was reconstituted with a complete mixture of 30S proteins to yield 30S ribosomes. Modified in vitro reconstitution conditions were required to obtain assembly of all of the synthetic ribosomes. Quantitative HPLC analysis of the protein content of each mutant showed that all of the proteins were present. The ability of synthetic 30S to form 70S particles under functional assay conditions was about 75% that of natural 30S and was unchanged by any of the mutations except for the deletion of G1401, which decreased the association activity under the standard conditions to 35-40% of synthetic 30S. That part of the ribosomal P site which interacts with the anticodon loop tRNA was investigated by near-UV (>300 nm) induced cross-linking of AcVal-tRNA. Cross-linking depended on both 30S subunits and the correct codon. The cross-linking yield of all mutants with a pyrimidine at position 1400 was equal to control isolated 30S, and the first-order rate constants for cross-linking of those mutants tested were like reconstituted natural 30S. The site of cross-linking for mutants with a C or U insertion between C1400 and G1401 was shifted to the inserted residue. Cross-linking to the base 5'' to G1401 rather than to the residue 3'' to C1399 indicates that G1401 is an important structural determinant of the P site.This publication has 21 references indexed in Scilit:
- Functional conservation near the 3' end of eukaryotic small subunit RNA: photochemical crosslinking of P site-bound acetylvalyl-tRNA to 18S RNA of yeast ribosomes.Proceedings of the National Academy of Sciences, 1982
- A gas-liquid solid phase peptide and protein sequenator.Journal of Biological Chemistry, 1981
- Correct codon-anticoden base pairing at the 5'-anticodon position blocks covalent crosslinking between transfer ribonucleic acid and 16S RNA at the ribosomal P siteBiochemistry, 1981
- Evidence for pyrimidine-pyrimidine cyclobutane dimer formation in the covalent cross linking between transfer ribonucleic acid and 16S ribonucleic acid at the ribosomal P siteBiochemistry, 1980
- Covalent crosslinking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acidBiochemistry, 1979
- A method to identily individual proteins in four different two-dimensional gel electrophoresis systems: Application to Escherichia coli ribosomal proteinsAnalytical Biochemistry, 1979
- Photochemical crosslinking unmodified acetylvalyl-tRNA to 16S RNA at the ribosomal P siteBiochemistry, 1978
- Formation of a thymine photoproduct in transforming DNA by near ultraviolet irradiationBiochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1977
- Structure and function of E. coli ribosomes. V. Reconstitution of functionally active 30S ribosomal particles from RNA and proteins.Proceedings of the National Academy of Sciences, 1968
- Peptides Derived from Tryptic Digestion of Egg White LysozymeJournal of Biological Chemistry, 1963