Abstract The approximate number and molecular weight of pea seedling ribosomal proteins were determined by two-dimensional electrophoresis on polyacrylamide gels containing either urea or sodium dodecyl sulfate. The small (40 S) ribosomal subunit contained 32 to 40 proteins and the large (60 S) subunit 44 to 55 proteins. The great majority of ribosomal proteins were basic and had molecular weights between 20,000 and 30,000. At least two proteins of higher molecular weight were found in the large subunit. Structural homologies between ribosomal proteins of different origin were estimated by two-dimensional electrophoretic analysis, by immunoelectrophoresis, double-diffusion tests, and quantitative immunoprecipitation. A high degree of evolutionary conservation was found, by the above methods, among ribosomal proteins of several species of higher plants, whereas little homology was found between chloroplast proteins and cytoplasmic ribosomal proteins of the same plant. No homologies were detected between chloroplast ribosomal proteins and proteins extracted from ribosomes of either mitochondria, bacteria, or blue-green algae.
The three-dimensional locations of Escherichia coli ribosomal proteins S13, L1, and L7/L12 on the surface of ribosomal subunits and 70S monomeric ribosomes were determined by electron microscopy of antibody-labeled ribosomal particles. A new approach to orient the subunits within 70S ribosomes was developed that used 30S.70S.50S triples that were prepared by simultaneous combination with one antibody directed against a 30S protein and another directed against a 50S protein. Electron microscope studies of triples obtained with the antibody combinations anti-S13/anti-L1 and anti-S13/anti-L7/L12 showed that, in 70S monomeric ribosomes, the head of the 30S subunit is proximate to protein L1 and the peptidyl transferase center but far from the rod-like appendage containing proteins L7 and L12.
Treatment of E. coli ribosomal subunits with 2-iminothiolane coupled with mild ultraviolet irradiation leads to the formation of a large number of RNA-protein cross-links. In the case of the 30S subunit, a number of sites on 16S RNA that are cross-linked to proteins S7 and S8 by this procedure have aLready been identified (see ref. 6). Here, by using new or modified techniques for the partial digestion of the RNA and the subsequent isolation of the cross-linked RNA-protein complexes, three new iminothiolane cross-links have been localized: Protein S17 is cross-linked to the 16S RNA within an oligonucleotide encompassing positions 629–633, and protein S21 is cross-linked to two sites within oligonucleotides encompassing positions 723–724 and positions 1531–1542 (the 3′-end of the 16S RNA).
By using immunoelectron microscopy, we have localized the binding site on 50S Escherichia coli ribosomal subunits for puromycin, an antibiotic that interacts with the ribosomal peptidyltransferase center. This was achieved by affinity-labeling 50S subunits with N-bromoacetyl puromycin and treating the labeled subunits with an antibody specific for the N6,N6-dimethyladenosine moiety of puromycin. The position of the puromycin binding site was then revealed by localizing the attachment sites of the IgG molecules on the surfaces of the 50S subunits under the electron microscope: it was located at the interface between the subunits, on and around the wider lateral protuberance of the 50S subunit. This localizes directly the peptidyl transferase center on the surface of the large ribosomal subunit.
'/%3e%3cuse xlink:href='%23a' transform='rotate(72 0 0)'/%3e%3cuse xlink:href='%23a' transform='rotate(-72 0 0)'/%3e%3cuse xlink:href='%23a' transform='scale(-1 1) rotate(72)'/%3e%3c/g%3e%3c/defs%3e%3cpath fill='%23012169' d='M0 0h1200v600H0z'/%3e%3cpath stroke='%23FFF' stroke-width='60' d='m0 0 600 300M0 300 600 0' clip-path='url(%23b)'/%3e%3cpath stroke='%23C8102E' stroke-width='40' d='m0 0 600 300M0 300 600 0' clip-path='url(%23c)'/%3e%3cpath stroke='%23FFF' stroke-width='100' d='M300 0v300M0 150h600' clip-path='url(%23b)'/%3e%3cpath stroke='%23C8102E' stroke-width='60' d='M300 0v300M0 150h600' clip-path='url(%23b)'/%3e%3cuse xlink:href='%23d' fill='%23FFF' transform='matrix(45.4 0 0 45.4 900 120)'/%3e%3cuse xlink:href='%23d' fill='%23C8102E' transform='matrix(30 0 0 30 900 120)'/%3e%3cg transform='rotate(82 900 240)'%3e%3cuse xlink:href='%23d' fill='%23FFF' transform='rotate(-82 519.022 -457.666) scale(40.4)'/%3e%3cuse xlink:href='%23d' fill='%23C8102E' transform='rotate(-82 519.022 -457.666) scale(25)'/%3e%3c/g%3e%3cg transform='rotate(82 900 240)'%3e%3cuse xlink:href='%23d' fill='%23FFF' transform='rotate(-82 668.57 -327.666) scale(45.4)'/%3e%3cuse xlink:href='%23d' fill='%23C8102E' transform='rotate(-82 668.57 -327.666) scale(30)'/%3e%3c/g%3e%3cuse xlink:href='%23d' fill='%23FFF' transform='matrix(50.4 0 0 50.4 900 480)'/%3e%3cuse xlink:href='%23d' fill='%23C8102E' transform='matrix(35 0 0 35 900 480)'/%3e%3c/svg%3e)