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Chapter: Genetics and Molecular Biology: Biological Assembly, Ribosomes and Lambda Phage

Determining Details of Local Ribosomal Structure

Consider the fundamental question of determining which proteins are close neighbors in the ribosome.

Determining Details of Local Ribosomal Structure

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 assem-bled, 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 ar-rangement 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 (Fig. 21.8).

In vitro assembly of ribosomes can be used to construct a ribosomein which two of the proteins contain the fluorescent probes. By illumi-nating the rebuilt ribosomes with light in the excitation spectrum of the

Figure 21.8 Spectra used in measuring distances separating ribosomal pro-teins. 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.

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 be-tween ribosomal proteins. This method has yielded the most informa-tion and the most reliable information on ribosome structure. It too relies on reassembly of ribosomal subunits. Two proteins in the ribo-some 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 inter-ference 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 consis-tent picture for the locations within the ribosomal subunits of the ribosomal proteins.

Figure 21.9 Technique for footprinting rRNA in the intact ribosome. Theenhanced 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.

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 transcrip-tase is hybridized. The elongation by reverse transcriptase ends at the modified bases, and the locations of protected bases can be determined (Fig. 21.9).

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