Interaction of the Bacillus stearothermophilus ribosomal protein S15 with 16 S rRNA: II. Specificity determinants of RNA-protein recognition.
S15 is a primary ribosomal protein that interacts specifically with a three-way junction in the central domain of 16 S rRNA, whose binding induces a conformational change in the RNA. In the accompanying paper, we demonstrated that S15 binds with high affinity to a 61 nucleotide RNA corresponding to the minimal rRNA binding site. Here, the sequence and structural determinants for the RNA in the Bacillus stearothermophilus S15-rRNA interaction have been probed using site-directed mutagenesis, chemical modification interference, and iodine footprinting of phosphorothioate RNA. Mutations and RNA modifications that interfere with protein binding cluster in two distinct regions, one containing an internal loop and the other containing a three-way junction. The internal loop, defined by two A.G base-pairs and a bulged guanosine, is not important for the specific interaction, however, BS15 interacts with a phylogenetically conserved G.U base-pair above this internal loop. Near the three-way junction in helix 22, a bulged adenosine and two base-pairs adjacent to the junction also provide important determinants for BS15 binding. Chemical modification interference also suggests that four highly phylogenetically conserved nucleotides in the three-way junction may form non-canonical G.G and U.A base-pairs that are required for the BS15-rRNA interaction. Ethylation modification interference suggests that BS15 binding is accompanied by a conformational change in the RNA involving orientation of helices 20 and 22 at an acute angle with respect to one another. Projection of the data provided by mutagenesis, chemical modification interference analysis, and iodine footprinting onto a three-dimensional model illustrates that BS15 is likely to interact with the minor groove along an extended face of helix 22.
Batey
R T
RT
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Williamson
J R
JR
D001483:Base Sequence / Q000235:genetics; D004047:Diethyl Pyrocarbonate; D005038:Ethylnitrosourea; D001411:Geobacillus stearothermophilus / Q000737:chemistry*; D006834:Hydrazines; D007455:Iodine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D013873:Thionucleotides
D006834:Hydrazines; D009711:Nucleotides; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins; D013873:Thionucleotides; C036004:ribosomal protein S15; C029424:hydrazine; D007455:Iodine; D004047:Diethyl Pyrocarbonate; D005038:Ethylnitrosourea