Messenger RNA recognition by fragments of ribosomal protein S4.
Ribosomal protein S4 from Escherichia coli binds a large domain of 16 S ribosomal RNA and also a pseudoknot structure in the alpha 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 full-length 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.
Baker
A M
AM
Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Draper
D E
DE
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000378:metabolism*; D015870:Gene Expression; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D010446:Peptide Fragments; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C031798:ribosomal protein S4; D008239:Lysine