Consider the fundamental question of determining which proteins are close neighbors in the ribosome. One direct approach to this question is to crosslink two proteins on the intact ribosome with bifunctional crosslinking reagents. If two ribosomal proteins are connected by the reagent when they are in a ribosome, but not when they are free in solution, it can be concluded that the proteins are near one another in the ribosome. This technique is fraught with artifacts, however, and results from different laboratories frequently do not agree, leading some to believe only those crosslinking results that have been duplicated in more than two laboratories.

Many of the proteins that are crosslinked to each other are proteins that depend on one another during assembly of the ribosomal subunit. A few of these proteins are encoded in the same operons. In one case, proteins that are adjacent to one another in the ribosome derive from adjacent genes in the chromosome. For example, ribosomal proteins S4, S11, and S13 lie in the same operon, S13-S11-S4. S4 and S13 and S13 and S11 crosslink, S4 and S13 interact during assembly, and together they interact with S11 during assembly.

The ability to reassemble ribosomes from their isolated components greatly facilitates structural studies. A ribosome can be partially assembled, for example, and then antibody against a component in the immature ribosome can be added. If the presence of the antibody blocks the subsequent association of a ribosomal protein added later, it is reasonable to expect that the antibody directly blocks access of the protein to its site.

If all ribosomal proteins were spherical, their complete spatial arrangement would be determined by knowing the distances between the centers of proteins. Some of the requisite measurements can be made with fluorescence techniques or slow neutron scattering. Fluorescent molecules possess an absorption spectrum such that illumination by photons within this wavelength band excites the molecule, which then emits a photon of longer wavelength within what is called the emission spectrum of the molecule (Fig1).

Fig1. Spectra used in measuring distances separating ribosomal proteins. Dotted line is the excitation and emission spectrum of fluorescent molecule 1 and the solid line is the excitation and emission spectra for molecule 2.

In vitro assembly of ribosomes can be used to construct a ribosome in which two of the proteins contain the fluorescent probes. By illuminating the rebuilt ribosomes with light in the excitation spectrum of the first molecule and measuring the strength of the fluorescence in the wavelength of the emission spectrum of the second molecule, the distance between the two fluorescent molecules can be determined. The amount of light in the second emission spectrum varies as the sixth power of the distance separating the molecules:

where R is the distance between the fluorescent molecules and Ro is a constant that depends on the orientations of the molecules, the spectral overlap of the fluorescent emission and excitation spectra, and the index of refraction of the medium separating the molecules. The method yields the most reliable data for proteins separated by 25 to 75 Å; that is, the method is best at determining the distances of nearest neighbors in the ribosome.

Neutron diffraction is another method of measuring distances between ribosomal proteins. This method has yielded the most information and the most reliable information on ribosome structure. It too relies on reassembly of ribosomal subunits. Two proteins in the ribosome are replaced by their deuterated equivalents. These proteins are obtained from cells grown on deuterated medium. Since the neutron scattering properties of hydrogen and deuterium are different, an interference pattern is generated by the presence in the ribosome of the two proteins with different scattering properties. The angular separation in the peaks of the interference pattern can be related to the distance separating the two altered proteins in the reconstituted ribosome. Overall, the results of crosslinking, assembly cooperativity, immune microscopy, fluorescence transfer, and neutron scattering give a consistent picture for the locations within the ribosomal subunits of the ribosomal proteins.

Ribosomal RNA can also be footprinted like DNA. Either bare RNA, RNA with a few proteins bound, or even an intact ribosome with or without a bound protein synthesis inhibitor like streptomycin can be used. The RNA is treated with chemicals like dimethylsulfate or kethoxal that react with unprotected nucleotides. Then the RNA is purified and a DNA oligonucleotide that will serve as a primer for reverse transcriptase is hybridized. The elongation by reverse transcriptase ends at the modified bases, and the locations of protected bases can be determined (Fig. 2).

Fig2. Technique for footprinting rRNA in the intact ribosome. The enhanced bands correspond to exposed bases. Normally a control would be done reacting denatured rRNA and rRNA that is in an intact ribosome. Then the bases protected and not protected are revealed by comparing the band intensities from the free RNA and the RNA from the ribosomes.

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مواضيع ذات صلة

Lambda Phage Assembly: Packaging the DNA and Formation of the cos Ends

Lambda Phage Assembly: The Head Assembly Sequence and Host Proteins

Lambda Phage Assembly: The Geometry of Capsids

Experiments with in vitro Ribosome Assembly

The Global Structure of Ribosomes

RNAse and Ribosomes

Assaying for Sequence Requirements of Gene Rearrangements

The D Regions in H Chains

The J Regions

Many Copies of V Genes and Only a Few C Genes

P Element Hopping by Chromosome Rescue

P Elements and Transformation