Interaction of the Bacillus stearothermophilus ribosomal protein S15 with its 5'-translational operator mRNA.
The Bacillus stearothermophilus ribosomal protein S15 (BS15) binds both a three-helix junction in the central domain of 16 S ribosomal RNA and its cognate mRNA. Native gel mobility-shift assays show that BS15 interacts specifically and with high affinity to the 5'-untranslated region (5'-UTR) of this cognate mRNA with an apparent dissociation constant of 3(+/-0.3) nM. In order to localize the structural elements that are essential for BS15 recognition, a series of deletion mutants of the full cognate mRNA were prepared and tested in the same gel-shift assay. The minimal binding site for BS15 is a 50 nucleotide RNA showing a close secondary structure resemblance to the BS15 binding region from 16 S rRNA. There are two major structural motifs that must be maintained for high-affinity binding. The first being a purine-rich three-helix junction, and the second being an internal loop. The sequence identity of the internal loops differs greatly between the BS15 mRNA and rRNA sites, and this difference is correlated to discrimination between wild-type BS15 and a BS15(H45R) mutant. The association and dissociation kinetics measured for the 5'-UTR-BS15 interaction are quite slow, but are typical for a ribosomal protein-RNA interaction. The BS15 mRNA and 16 S rRNA binding sites share a common secondary structure yet have little sequence identity. The mRNA and rRNA may in fact present similar if not identical structural elements that confer BS15 recognition.
Scott
L G
LG
Department of Molecular Biology and Skaggs Institute for Chemical Biology, MB33, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Williamson
J R
JR
D020121:5' Untranslated Regions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D024202:Electrophoretic Mobility Shift Assay; D001411:Geobacillus stearothermophilus / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D017186:Titrimetry
D020121:5' Untranslated Regions; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C036004:ribosomal protein S15