Chemical footprinting

Translocation occurs slowly even in the absence of GTP. However, it is greatly enhanced by GTP hydrolysis.3943 402 Even empty ribosome, without tRNAs, but in the presence of GTP and EF-Tu and EF-G, hydrolyze GTP. The ribosome may sequentially bind EF-Tu, then EF-G, oscillating between two differing states.403 Tire movement of mRNA through the ribosome has been plotted using a variety of immunochemical, crosslinking, and chemical footprinting methods.52 404-407... 

To place a dye at internal positions without breaking the RNA backbone, the Pan lab developed a method that involves replacing nonessential hairpin loops within RNA sequences with larger loops with specific sequences that are hybridized to labeled DNA oligos (Smith et al., 2005). These modifications had little effect on structure, as assayed by chemical footprinting, or catalytic efficiency of the catalytic domain ofRNase P. The same method was successfully employed for studying ribosome dynamics (Dorywalska et al., 2005). 

Extensive study has established that the ion atmosphere is a diffuse and dynamic structure (Misra and Draper, 2000) that cannot be described by simple ion-binding models (Manning, 1969a,b,c). This means that structural techniques such as crystallography or chemical footprinting cannot be used to study the ion atmosphere. Therefore, the techniques available for studying the ion atmosphere are severely restricted. 

Fluorescence assay (BiFC) Circular dlchroism spectroscopy Enzymatic chemical footprinting... 

Figure 9.2. (A) Secondary structure proposed previously for the citrulline- and arginine-specific aptamers, based on co-variations of selected sequences, on the chemical footprinting pattern obtained in the presence of the cognate amino acid, as well as in damage selection experiments. The bases which were conserved among different isolates arc shown in upper case variant bases are in lower case. The three nucleotides critical for arginine specificity (13,29 and 31) arc indicated by circles (for citrulline) and boxes (for arginine). (B) Tertiary structure of the L-arginine aptamer complex resolved by NMR spectroscopy. Yellow L-arginine red the three mutations. (Illustration adapted from [9].)...

Several metal complexes now serve as high-resolution, sequence-neutral chemical footprinting reagents. Some of these reagents are shown in Figure 8.13. The first, as mentioned previously, was MPE-Fe(II). The complex con-... 

Other transition-metal complexes are also finding applications in chemical footprinting. Both Cu(phen)2" and manganese porphyrins have been used to footprint DNA-binding proteins.These complexes likely cleave DNA through... 

RNAs, mutational analysis, neutron scatter-ing, chemical and photochemical crosslinking, photoaffinity labeling, immunological labeling, chemical footprinting, fluorescence resonance energy transfer (FRET), mass spectrometry, and study of the effects of toxic proteins (Box 29-A) and antibiotics (Box 29-B). 

The binding of photolyase to its substrate has been extensively investigated with DNase and chemical footprinting methods (Baer and Sancar, 1989 Husain et al, 1987 Kiener et al, 1989) and with substrates ranging in... 

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