FUNCTIONAL-EFFECTS OF BASE CHANGES WHICH FURTHER DEFINE THE DECODING CENTER OF ESCHERICHIA-COLI 16S RIBOSOMAL-RNA - MUTATION OF C1404, G1405, C1496, G1497, AND U1498
Pr. Cunningham et al., FUNCTIONAL-EFFECTS OF BASE CHANGES WHICH FURTHER DEFINE THE DECODING CENTER OF ESCHERICHIA-COLI 16S RIBOSOMAL-RNA - MUTATION OF C1404, G1405, C1496, G1497, AND U1498, Biochemistry, 32(28), 1993, pp. 7172-7180
The existence and functional importance of the tertiary base pair G140
1:C1501, which brings together two universally present and highly sequ
ence-conserved single-stranded segments of small subunit ribosomal RNA
, was proven recently by mutational analysis [Cunningham, P. R., Nurse
, K., Bakin, A., Weitzmann, C. J., Pflumm, M., & Ofengand, J. (1992) B
iochemistry 31, 12012-12022]. Here we show that the additional nearby
tertiary base pairs C1404:G1497 and G1405:C1496 also exist and are fun
ctionally important for tRNA binding to the ribosomal A and P sites. B
reakage of the base pairs in turn led to a loss of activity at both A
and P sites, whereas restoration in the reverse orientation led to rec
overy of activity. Recovery was incomplete, indicating that base pairi
ng alone is insufficient for full restoration of function. Mutation of
U1498 to G created the potential for the tertiary base pair C1403:G14
98, which could stack on the aforementioned double base pair, creating
a more stable helix longer by one residue. This mutation did not affe
ct subunit association, A- and P-site binding of tRNA to 70S, fMet-tRN
A binding to 30S, or poly(Phe) synthesis but did block formation of th
e first peptide bond, fMet-Val. Mutation of U1498 to A or C did not sh
ow this effect. Since the G1498 mutant could make both the 70S initiat
ion complex and the peptide bond, as shown by its ability to form fMet
-puromycin, the block in fMet-Val synthesis appears to involve some as
pect of A-site function. Codon recognition at the A site seemed normal
since the mutant did not miscode in an in vitro Leu-tRNA/poly(U) assa
y, although an effect on frameshifting was not ruled out. Codon-antico
don interaction at the P site also appeared normal as judged by cross-
linking of P-site-bound tRNA exclusively to C1400 at a normal rate and
yield.