Using a multicopy plasmid encoding the Escherichia coli rrnB ribosomal RNA operon and the techniques of in vitro site-directed mutagenesis, we have introduced several small alterations into the central domain of 16 S rRNA, which encompasses nucleotides 560 to 890. Four of the rRNAs studied contained deletions and one contained an insertion. The altered small ribosomal subunit rRNAs were used to investigate relationships among 16 S rRNA processing, protein-16 S rRNA interactions and assembly of the 30 S ribosomal subunit. Analysis of plasmid-coded transcripts from maxicells revealed that products from wild-type 16 S rRNA genes were fully processed and assembled into mature 30 S subunits. Under the same conditions, the processing and assembly of transcripts derived from the mutant plasmids were severely impaired. In some instances, the mutations completely blocked both processes, while in other cases rRNA maturation and ribosome assembly were retarded, but not eliminated completely. In all cases, the mutations led to the accumulation of the 17 S precursor to 16 S rRNA. The mutant 17 S rRNAs were purified and incubated with various combinations of E. coli ribosomal proteins S6, S8, S15 and S18, which are known to bind to the central domain of 16 S rRNA. Ribonuclease digestion of the resulting protein-17 S rRNA complexes and fractionation of the products permitted detection of three distinct protein-RNA fragment complexes which contained S8, S8 + S15, or S6 + S8 + S15 + S18. Whereas wild-type 17 S rRNA was able to form all three of these complexes, deletion of nucleotides 693 to 721 or 822 to 874 abolished the interaction of S6 and S18, and removal of nucleotides 659 to 718 prevented the binding of S6, S15 and S18. In contrast, elimination of residue 614, or the presence of a 16-base insertion between nucleotides 614 and 615, had no significant effect on the binding of any of the four proteins tested. Together, our results demonstrate that 16 S rRNA maturation and 30 S subunit assembly are tightly coupled, and show that, in at least some cases, defects in these processes can be correlated with the inability of particular ribosomal proteins to associate with altered rRNA molecules. Moreover, we have confirmed the essentiality of certain rRNA sequences for the formation and/or stabilization of these protein-rRNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)