Interaction of proteins S16, S17 and S20 with 16 S ribosomal RNA.

We have used rapid chemical probing methods to examine the effect of assembly of ribosomal proteins S16, S17 and S20 on the reactivity of individual residues of 16 S rRNA. Protein S17 strongly protects a compact region of the RNA between positions 245 and 281, a site previously assigned to binding of S20. Protein S20 also protects many of these same positions, albeit more weakly than S17. Strong S20-dependent protections are seen elsewhere in the 5' domain, most notably at positions 108, and in the 160-200 and 330 loop regions. Enenpectedly, S20 also causes protection of several bases in the 1430-1450 region, in the 3' minor domain. In the presence of the primary binding proteins S4, S8 and S20, we observe a variety of effects that result from assembly of the secondary binding protein S16. Most strongly protected are nucleotides around positions 50, 120, 300 to 330 and 360 in the 5' domain, and positions 606 to 630 in the central domain. In addition, numerous nucleotides in the 5' and central domains exhibit enhanced reactivity in response to S16. Interestingly, the strength of the S20-dependent effects in the 1430-1450 region is attenuated in the presence of S4 + S8 + S20, and restored in the presence of S4 + S8 + S20 + S16. Finally, the previously observed rearrangement of the 300 region stem-loop that occurs during assembly is shown to be an S16-dependent event. We discuss these findings with respect to assignment of RNA binding sites for these proteins, and in regard to the co-operativity of ribosome assembly.

[1]  B. Ballou,et al.  Assembly mapping of 30 S ribosomal proteins from Escherichia coli. Further studies. , 1974, The Journal of biological chemistry.

[2]  A. Kerlavage,et al.  Reversed-phase high-performance liquid chromatography of Escherichia coli ribosomal proteins. Characteristics of the separation of a complex protein mixture. , 1983, Journal of chromatography.

[3]  H. Noller,et al.  Localization of the binding site for protein S4 on 16 S ribosomal RNA by chemical and enzymatic probing and primer extension. , 1986, Journal of molecular biology.

[4]  R. Garrett,et al.  The topography of the 5' end of 16-S RNA in the presence and absence of ribosomal proteins S4 and S20. , 1980, European journal of biochemistry.

[5]  L. Changchien,et al.  Studies on the role of amino acid residues 31 through 46 of ribosomal protein S4 in the mechanism of 30 S ribosome assembly. , 1978, Journal of molecular biology.

[6]  M. Nomura,et al.  Assembly Mapping of 30S Ribosomal Proteins from E. coli , 1970, Nature.

[7]  H. Noller,et al.  Probing the assembly of the 3' major domain of 16 S ribosomal RNA. Quaternary interactions involving ribosomal proteins S7, S9 and S19. , 1988, Journal of molecular biology.

[8]  H. Noller.  Structure of ribosomal RNA. , 1984, Annual review of biochemistry.

[9]  C. Ehresmann,et al.  RNA-protein interactions in the ribosome. I. Characterization and ribonuclease digestion of 16 S RNA-ribosomal protein complexes. , 1974, Journal of molecular biology.

[10]  G. Mora,et al.  The ribosomal proteins of Escherichia coli. I. Purification of the 30S ribosomal proteins. , 1969, Biochemistry.

[11]  C. Kurland.  The requirements for specific sRNA binding by ribosomes. , 1966, Journal of molecular biology.

[12]  R. Gutell,et al.  Comparative anatomy of 16-S-like ribosomal RNA. , 1985, Progress in nucleic acid research and molecular biology.

[13]  J. Ebel,et al.  Characterization of the regions from E. coli 16 S RNA covalently linked to ribosomal proteins S4 and S20 after ultraviolet irradiation , 1977, FEBS letters.

[14]  R. Garrett,et al.  An investigation of the 16-S RNA binding sites of ribosomal proteins S4, S8, S15, and S20 FROM Escherichia coli. , 1975, European journal of biochemistry.

[15]  H. Noller,et al.  Rapid chemical probing of conformation in 16 S ribosomal RNA and 30 S ribosomal subunits using primer extension. , 1986, Journal of molecular biology.

[16]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[17]  R. Brimacombe,et al.  The localization of multiple sites on 16S RNA which are cross-linked to proteins S7 and S8 in Escherichia coli 30S ribosomal subunits by treatment with 2-iminothiolane. , 1983, Nucleic acids research.

[18]  M. Capel,et al.  Preparative ion-exchange high-performance liquid chromatography of bacterial ribosomal proteins. , 1986, Analytical biochemistry.

[19]  C. Kurland,et al.  Stoichiometry of the 30S ribosomal proteins of Escherichia coli. , 1971, Biochemistry.

[20]  G. Mackie,et al.  RNA--protein interactions in the ribosome. IV. Structure and properties of binding sites for proteins S4, S16/S17 and S20 in the 16S RNA. , 1978, Journal of molecular biology.

[21]  H. Noller,et al.  Interaction of ribosomal proteins, S6, S8, S15 and S18 with the central domain of 16 S ribosomal RNA. , 1988, Journal of molecular biology.

[22]  E. Eikenberry,et al.  Separation of large quantities of ribosomal subunits by zonal ultracentrifugation. , 1970, European journal of biochemistry.

[23]  R. Brimacombe,et al.  RNA-protein cross-linking in Escherichia coli ribosomal subunits: localization of sites on 16S RNA which are cross-linked to proteins S17 and S21 by treatment with 2-iminothiolane. , 1986, Nucleic acids research.