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PubMed1986

Negatively stained 50 S ribosomal subunits of Escherichia coli.

Ribosomes are large nucleoproteins of approximately 3 X 10(6) Mr. In contrast to helical or spherical nucleoproteins (viruses) of similar size (which consist of several hundred small asymmetric units reproduced by symmetry), ribosomes are completely asymmetric; therefore, the amount of structural information (defined by the number of independent image elements) necessarily increases from about 10 to 20 to about 1000 to 2000 (at resolutions of the order of 2 nm). With present techniques, only stained single particles can be studied in the electron microscope. Our published work on the 30 S subunit and on the 50 S subunit has demonstrated that three-dimensional reconstructions of stained single particles show a great number of structural details that are reproducible if the particles have the same orientation. One of the main results of this paper is the final proof of this reproducibility from detailed comparisons of individual 50 S subunits and of independent averages over a few (3 to 5) particles in the kidney or crown orientation; in the latter case, even after a chemical modification. The 50 S subunit is non-uniformly stained along the optical axis. It displays a complicated, stain-filled channel-like structure, within which is approximately the partial volume expected for the RNA. The particle shows an irregular but reproducible boundary surface against the stain. At several sites, the channel structure protrudes to the surface. Since the secondary structure of the RNA is well known, one might try to locate it in the subunit after chemical identification of its surface contacts (the 3' end of 23 S RNA and the 3' end of the 5 S RNA have been localized). Most interesting is a groove on the surface, which might accommodate the mRNA.

Computer Simulation↗
PubMed1986

Fibers of RecA protein and complexes of RecA protein and single-stranded phi X174 DNA as visualized by negative-stain electron microscopy.

Monomers of purified RecA protein polymerize into helical fibers whose pitch is 7.2 nm to 7.5 nm and whose diameter is 11 nm. Either short (approximately 0.2 micron), single fibers, or bundles of aligned, longer fibers, can be formed preferentially, by varying the Mg2+ concentration. When RecA protein is bound to circular, single-stranded phi X174 DNA it forms helical fibers of different classes of contour lengths, ranging from 0.98 micron, depending upon the conditions of assembly. Two different helical pitches are found, one of 9.3 nm when the incubation buffer contains, besides the obligatory Mg2+, either ATP gamma S or ATP accompanied by single-strand binding protein, and one of 5.5 nm when the latter additives are omitted. Preformed fibers of the compact type can be converted to open ones of 9.3 nm pitch upon addition of ATP gamma S, even after the removal of unbound RecA. All signs of helicity are obliterated upon glutaraldehyde cross-linking except in those fibers whose assembly has been mediated by ATP gamma S. RecA protein and single-strand binding protein are competitively bound to single-stranded DNA. Composite complexes, however, are not encountered unless ATP gamma S is present. Otherwise, segments of DNA that are coated by one or the other protein are seen as separate regions. When the assembly of complexes of single-stranded DNA and RecA is mediated by single-strand binding protein and ATP, the axial separation between successive bases is 0 X 42 nm, somewhat greater than the axial distance between bases in one strand of duplex DNA in the B form. It is proposed that the bases of the single-stranded DNA in the complex are located near its inner surface, and that base-pairing with double-stranded DNA takes place following invasion of the central cavity of the complex.

Adenosine Triphosphate↗