In vitro assembly of 30S and 70S bacterial ribosomes from 16S RNA containing single base substitutions, insertions, and deletions around the decoding site (C1400)

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 mutant 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 of tRNA was investigated by near-UV (greater than 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.