TY - JOUR
T1 - Isolation of ribosomal protein-RNA complexes by nitrocellulose membrane filtration
T2 - Equilibrium binding studies
AU - Spicer, Eleanor
AU - Schwarzbauer, Jean
AU - Craven, Gary R.
N1 - Funding Information:
In summary we have developed a rapid and convenient technique which permits the accurate determination of association constants between ribosomal proteins and rRNA. This approach has numerous potential applications. For example, many Important parameters such as temperature, ionic conditions, and solvent conditions can be quantitatively analyzed for their effect on protein-RNA recognition. In addition, ribosomal proteins which have not previously been shown to interact with rRNA can be surveyed for possible specific associations too weak to measure by the traditional techniques. ACKNOWLEDGEMENTS This work was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison and by research grant GM 15422 from the National Institutes of Health. We appreciate the use of the pilot plant operated by the Biochemistry Department, supported by U.S. Public Health Service grant Fr-00214. We thank Cathy Bloomer for the preparation of purified 30S proteins. During the course of this work E.S. was supported by N.I.H. Training Grant GM1O874.
PY - 1977/2
Y1 - 1977/2
N2 - E. coli ribosomal proteins are retained by nitrocellulose filters. In contrast, 16S RNA passes through nitrocellulose filters. We have found that specific protein-RNA complexes involving single proteins also pass through nitrocellulose filters. Thus, by utilizing radioactively labeled r-proteins, nitrocellulose filtration can be used to study directly and sensitively the stoichiometry of r-protein-RNA association. The filtration process maintains near equilibrium conditions1, making it applicable to weak as well as strong protein-RNA associations.We have used nitrocellulose filtration to obtain saturation binding curves for the association of proteins S4, S7, S8 and S20 with 16S RNA. In each case, the stoichiometry of binding was one mole of protein or less per mole of RNA. The stoichiometry of protein S8 binding to 16S RNA measured by filtration is comparable to that observed by sucrose gradient centrifugation. Association constants for the binding of proteins S4, S8 and S20 to 16S RNA have been determined by analysis of the saturation binding curves and were found to range from .3-6 × 107 M-1
AB - E. coli ribosomal proteins are retained by nitrocellulose filters. In contrast, 16S RNA passes through nitrocellulose filters. We have found that specific protein-RNA complexes involving single proteins also pass through nitrocellulose filters. Thus, by utilizing radioactively labeled r-proteins, nitrocellulose filtration can be used to study directly and sensitively the stoichiometry of r-protein-RNA association. The filtration process maintains near equilibrium conditions1, making it applicable to weak as well as strong protein-RNA associations.We have used nitrocellulose filtration to obtain saturation binding curves for the association of proteins S4, S7, S8 and S20 with 16S RNA. In each case, the stoichiometry of binding was one mole of protein or less per mole of RNA. The stoichiometry of protein S8 binding to 16S RNA measured by filtration is comparable to that observed by sucrose gradient centrifugation. Association constants for the binding of proteins S4, S8 and S20 to 16S RNA have been determined by analysis of the saturation binding curves and were found to range from .3-6 × 107 M-1
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U2 - 10.1093/nar/4.2.491
DO - 10.1093/nar/4.2.491
M3 - Article
C2 - 320562
AN - SCOPUS:0017626329
SN - 0305-1048
VL - 4
SP - 491
EP - 499
JO - Nucleic acids research
JF - Nucleic acids research
IS - 2
ER -