Potential contact sites between the protein and RNA subunit in the Bacillus subtilis RNase P holoenzyme.
We have detected by nucleotide analog interference mapping (NAIM) AMPalphaS and IMPalphaS modifications in Bacillus subtilis RNase P RNA that interfere with binding of the homologous protein subunit. Interference as well as some enhancement effects were clustered in two main areas, in P10.1a/L10.1 and P12 of the specificity domain (cluster 1, domain I) and in P2, P3, P15.1, J18/2 and J19/4 of the catalytic domain (cluster 2a, domain II). Minor interferences in P1 and P19 and a strong and weak enhancement effect in P19 represent a third area located in domain II (cluster 2b). Our results suggest that P3, P2-J18/2 and J19/4 are key elements for anchoring of the protein to the catalytic domain close to the scissile phosphodiester in enzyme-substrate complexes. Sites of interference or enhancement in clusters 1 and 2a are located at distances between 65 and 130 A from each other in the current 3D model of a full-length RNase P RNA-substrate complex. Taking into account that the RNase P protein monomer can bridge a maximum distance of about 40 A, simultaneous direct contacts to the two aforementioned potential RNA-binding areas would be incompatible with our current understanding of bacterial RNase P RNA architecture. Our findings suggest that the current 3D model has to be rearranged in order to reduce the distance between clusters 1 and 2a. Alternatively, based on the recent finding that B. subtilis RNase P forms a tetramer consisting of two protein and two RNA subunits, cluster 1 may reflect one protein contact site in domain I, and cluster 2a a separate one in domain II.
Rox
Christoph
C
Institut für Biochemie, Medizinische Universität zu Lübeck, Ratzeburger Allee 160, Lübeck, D-23538, Germany.
Feltens
Ralph
R
Pfeiffer
Thomas
T
Hartmann
Roland K
RK
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D004492:Edetic Acid / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007455:Iodine / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009942:Organometallic Compounds / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043262:Ribonuclease P; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D005296:Ferrous Compounds; D020035:Holoenzymes; D009942:Organometallic Compounds; D021122:Protein Subunits; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; C055526:Fe(II)-EDTA; C088321:nickel nitrilotriacetic acid; D007455:Iodine; D004492:Edetic Acid; D004722:Endoribonucleases; D043262:Ribonuclease P; D009571:Nitrilotriacetic Acid