Incorporation of single dinitrophenyl-modified proteins in to the 30S subunit of Escherichia coli ribosomes by total reconstitution for localization by immune electron microscopy

Thesis/Dissertation · Sun Jan 01 04:00:00 EST 1989

OSTI ID:6159165

Olah, T V

The ribosome is a structurally defined organelle whose function is central to the existence of all organisms. It is the unique site of protein biosynthesis in all cells. A detailed understanding of ribosome structure is essential in understanding the process of translation. This thesis represents a new approach to the systematic localization of individual proteins contained in the small subunit of Escherichia coli ribosomes using immunoelectron microscopy. All 30S proteins were purified using high performance liquid chromatography (HPLC) and eight isolated proteins (S12,S21,S14,S19,S18,S17,S16 and S13) were derivatized with 2,4-(3,5-{sup 3}H)dinitrofluorobenzene (DNFB). The extent of modification of these proteins was estimated by both radioactivity and integrated peak areas, using dual wavelength monitoring at 214nm to detect protein and 360nm (to detect dinitrophenyl groups). Each dinitrophenylated protein was introduced in place of the corresponding unmodified protein into totally reconstituted 30S subunits. Antibodies raised against the DNP-hapten bound effectively to such reconstituted subunits and did not cause dissociation of the modified protein from the subunit. Electron microscopy of the immune complexes was used to localize the modified protein on the subunit surface. Incorporation of any of the DNP-modified proteins, with the exception of DNP-S18, does not interfere with the functionality of the ribosome as measure by the binding of Phe-tRNA{sup Phe} or the synthesis of poly(Phe) in a poly(U)-dependent manner. Results show that unmodified protein competes with DNP-protein and that DNP-protein can function, as its native counterpart, in stimulating uptake of specific proteins during reconstitution. This data provides evidence that each DNP-protein occupies the same position in 30S subunits as does the corresponding unmodified protein.

• Research Organization: Pennsylvania Univ., Philadelphia, PA (USA)
• OSTI ID: 6159165
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANTIBODIES
BACTERIA
BIOLOGICAL FUNCTIONS
BIOLOGICAL LOCALIZATION
BIOSYNTHESIS
CELL CONSTITUENTS
CHROMATOGRAPHY
ELECTRON MICROSCOPY
ESCHERICHIA COLI
FUNCTIONS
HYDROGEN COMPOUNDS
IMMUNE REACTIONS
ISOTOPE APPLICATIONS
LIQUID COLUMN CHROMATOGRAPHY
MICROORGANISMS
MICROSCOPY
NITRO COMPOUNDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
PROTEINS
RIBOSOMES
SEPARATION PROCESSES
SYNTHESIS
TRACER TECHNIQUES
TRITIUM COMPOUNDS