Ribosomal protein S4 from Escherichia coli binds a large domain of 16 S ribosomal RNA and also a pseudoknot structure in the ␣ operon mRNA, where it represses its own synthesis. No similarity between the two RNA binding sites has been detected. To find out whether separate protein regions are responsible for rRNA and mRNA recognition, proteins with N-terminal or C-terminal deletions have been overexpressed and purified. Protein-mRNA interactions were detected by (i) a nitrocellulose filter binding assay, (ii) inhibition of primer extension by reverse transcriptase, and (iii) a gel shift assay. Circular dichroism spectra were taken to determine whether the proteins adopted stable secondary structures. From these studies it is concluded that amino acids 48 -104 make specific contacts with the mRNA, although residues 105-177 (out of 205) are required to observe the same toeprint pattern as fulllength protein and may stabilize a specific portion of the mRNA structure. These results parallel ribosomal RNA binding properties of similar fragments (Conrad, R. C., and Craven, G. R. (1987) Nucleic Acids Res. 15, 10331-10343, and references therein). It appears that the same protein domain is responsible for both mRNA and rRNA binding activities.Functional studies of ribosomes have tended to focus on the roles of the ribosomal RNAs in recent years, as a number of studies have uncovered specific contributions of different rRNA domains to ribosome activities (1). As more ribosomal protein sequences have become available, it is becoming clear that a number of these proteins are highly conserved among all organisms and must also have specific and necessary roles in ribosome function. An intriguing set of ribosomal proteins are those that bind directly and independently to the ribosomal RNAs and also autogeneously regulate ribosomal protein expression. In many cases the regulation is due to the protein recognition of the mRNA translational initiation region (2), although protein binding to a pre-mRNA splice site has also been observed (3). These instances of a single protein carrying out two different RNA-related functions provide interesting systems for studying how RNA recognition has evolved and is related to specific protein functions (4).In several instances there is convincing similarity between the secondary structures of the mRNA and rRNA targets of an autoregulatory ribosomal protein (4 -6). It is reasonable to conclude that both mRNA and rRNA bind in the same active site of any one of these proteins. In other cases there is no obvious similarity between the two RNA substrates. For instance, the mRNA target site for Escherichia coli S4 protein is a complex pseudoknot of about 110 nucleotides within the ␣ operon mRNA (7). Nearly the entire 5Ј domain of the 16 S rRNA, a fragment of 460 nucleotides, is needed to form the ribosomal binding site for the protein (8), although a smaller region is protected from cleavage by bound protein (9). There is no primary or secondary structural similarity between the two target sites, ...