Incorporation of single dinitrophenyl-modified proteins into the 30 S subunit of Escherichia coli ribosomes by total reconstitution.
In this first of two consecutive papers, the main objective of which is to present a new approach to the systematic localization of individual proteins located in the Escherichia coli ribosome by immunoelectron microscopy, we describe the derivatization of several purified 30 S proteins (S12, S21, S14, S19, S18, S17) with 2,4-[3,5-3H]dinitrofluorobenzene at pH 7.4 and 8.4 and the uptake of each dinitrophenylated protein in place of the corresponding unmodified protein into totally reconstituted 30 S subunits. Reverse-phase high performance liquid chromatography is used to purify the proteins, to separate and characterize the products of 2,4-[3,5-3H]dinitrofluorobenzene modification, and to analyze the protein composition of the reconstituted subunits. The extent of dinitrophenyl (DNP) modification is estimated by both radioactivity and integrated peak areas, using dual wavelength monitoring at 214 and 360 nm. DNP derivatives of each of the six proteins are efficiently incorporated into reconstituting 30 S subunits. Incorporation of any of the six DNP-modified proteins does not interfere with binding of Phe-tRNA(Phe) in a poly(U)-dependent manner. This result, as well as data showing that unmodified protein competes with DNP-protein for uptake during reconstitution, provide evidence that each DNP-protein occupies the same position in 30 S subunit as does unmodified protein. In general, for a given protein, unmodified and/or less modified forms are incorporated in preference to more modified forms. Modification of protein S19 at pH 7.4 proceeds with selective formation of one derivative in high yield. Reverse-phase high performance liquid chromatography analysis of acid hydrolysates of a purified sample of this derivative, as well as of peptides derived from it by digestion with Staphylococcus aureus protease, show the N-terminal proline to be the predominant site of DNP-derivatization.
Olah
T V
TV
Department of Chemistry, University of Pennsylvania, Philadelphia 19104.
Olson
H M
HM
Glitz
D G
DG
Cooperman
B S
BS
D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004139:Dinitrofluorobenzene / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure; D009578:Nitrobenzenes / Q000494:pharmacology*; D011485:Protein Binding; D012269:Ribosomal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure; D013053:Spectrophotometry
D000596:Amino Acids; D009578:Nitrobenzenes; D012269:Ribosomal Proteins; D004139:Dinitrofluorobenzene