The existence and functional importance of the tertiary base pair G1401:C1501, which brings together two universally present and highly sequence-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) Biochemistry 31, 12012-12022]. Here we show that the additional nearby tertiary base pairs C1404:G1497 and G1405:C1496 also exist and are functionally important for tRNA binding to the ribosomal A and P sites. Breakage 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 recovery of activity. Recovery was incomplete, indicating that base pairing alone is insufficient for full restoration of function. Mutation of U1498 to G created the potential for the tertiary base pair C1403:G1498, which could stack on the aforementioned double base pair, creating a more stable helix longer by one residue. This mutation did not affect subunit association, A- and P-site binding of tRNA to 70S, fMet-tRNA binding to 30S, or poly(Phe) synthesis but did block formation of the first peptide bond, fMet-Val. Mutation of U1498 to A or C did not show this effect. Since the G1498 mutant could make both the 70S initiation complex and the peptide bond, as shown by its ability to form fMet-puromycin, the block in fMet-Val synthesis appears to involve some aspect of A-site function.(ABSTRACT TRUNCATED AT 250 WORDS)