Synthesis and Properties of N‐Acetyl‐N′‐(p‐glyoxylylbenzoyl)cystamine, a New Reagent for RNA‐RNA and RNA‐Protein Cross‐Linking

Abstract

The synthesis of N-acetyl-N'-(p-glyoxylylbenzoy1)cystamine (Gbz-Cynz-Ac) is described. Like other glyoxal-type reagents it reacts with guanine and arginine. The kinetics and pH dependence of these reactions are studied. (Gbz-Cynz-Ac) reacts with non-base-paired guanines at about 20 sites in 16-S rRNA. After reduction of disulfide bonds each derivatized guanine residue carries a free -SH group which can be used to create RNA-RNA bridges or, after introducing an additional photoactivatable derivative, RNA-protein bridges.Analysis of ribosome structure requires the development of methods for obtaining information about the conformation of RNA and proteins, and about the mechanisms and sites of the interactions between these molecules in the intact ribosome. A number of experimental procedures have been used for this purpose and have yielded a large amount of data concerning, in particular, the proximity relationships between ribosomal proteins [l -31. However few methods for studying RNA-protein interactions and RNA conformation in the ribosome are as yet available. Among those which have given valuable results the most productive are isolation and analysis of ribonucleoprotein fragments after ribonuclease digestion of ribosomes (proximity relationships between ribo- non-base-paired sequences in an RNA react with glyoxal derivatives, and the adducts formed (see Fig. 2) are stable at acid pH but unstable at alkaline pH. Cross-links introduced between adjacent unpaired regions of an RNA chain or between RNA and protein by the use of glyoxal-based reagents are therefore easily reversible. N-Acetyl-N'-(p-glyoxylylbenzoy1)-cystamine, whose synthesis and reactivity, are described can be used to introduce such cross-links in ribosomes or in free ribosomal RNA. Its use for the analysis of the structure of 16-S RNA and 30-S ribosomal subunits of Escherichia coli will be described in subsequent publications. Several other new reagents for RNA-protein cross-linking have recently been described [15 -171 and their use will probably contribute to the identification of the sites of interaction or of proximity of ribosomal RNAs and proteins.

Surface Modification Chemistry Based on the Electrostatic Adsorption of Poly-l-arginine onto Alkanethiol Modified Gold Surfaces

“…The reaction of glyoxal with the pArg layer (adsorbed from a borate buffer solution 0.1 M pH 9) was performed by soaking the pArg-terminated surface into 10 mM solution of the dicarbonyl reagent buffered at pH 9, 0.1 M borate buffer, for 4 h. In phenylglyoxal, the solvent was a 1:1 methanol/borate buffer mixture. It has been shown 37 that borate stimulates the kinetics of adduct formation of glyoxal compound derivatives that both enhances the amount of adduct formation and suppresses the formation of diaddition products.…”