Affinity labeling of the ribosomal decoding site with an AUG-substrate analog.

Abstract

The trinucleotide AUG was condensed at the 5'-end with N-bromoacetyl-p-.aminophenylphosphate.This bromoacetylated AUG analog reacted irreversibly with the mRNA binding site of Escherichia coli 70S ribosomes. After reaction of 70S ribosomes with the AUG analog, labeled 30S subunits could be isolated that were programmed for initiation-factor-dependent binding of fMet-tRNAfMet. This shows that this AUG-affinity label reacted in the decoding site for fMet-tRNAfMet. By combination of sodium dodecyl sulfate-, Sarkosyl-, and ureapolyacrylamide gel electrophoresis the AUG-affinity label was found to be irreversibly bound to ribosomal proteins S4, the ram gene product, and S18.Despite recent advances in our understanding of the function of particular ribosomal proteins in protein synthesis (for a review see ref. 1), several important problems remain. Among these is the role of ribosomal proteins in decoding mRNA. This is placed in the perspective of the number of proteins necessary to initiate, propagate, and terminate translation and their function in moving along the message. One way of studying this essential aspect of the translation process is to identify the ribosomal proteins that are directly involved in this part of protein synthesis. Recently, the technique of affinity labeling was successfully applied in several laboratories in order to elucidate the ribosomal proteins that are implicated in the peptidyltransferase center (2-4). Likewise, affinity labeling should help to answer other questions on the functions of ribosomal proteins. We have synthesized a bromoacetylated AUG analog, which irreversibly reacts at the decoding site of Escherichia coli ribosomes. After chemical linkage of this AUG analog, 30S subunits could be isolated, which were programmed for initiationfactor-dependent binding of fMet-tRNAfMet. This allowed identification of ribosomal proteins associated with decoding of mRNA.MATERIALS AND METHODS Ribosomes of E. coli A19 were isolated and purified as described (5). They were salt-washed once with 1 M NH4Cl.About 60-70% of these ribosomes bound fMet-tRNAfMet in a puromycin-sensitive state and represented tight couples according to the nomenclature of Noll (6). fMet-tRNAfMet was prepared from purified E. coli tRNAfMet (Boehringer, Germany) according to ref. (pH 9.0) buffer. After treatment of the incubation mixture with alkaline phosphatase, nitrophenyl-pA-U was isolated by TEAE-cellulose column chromatography using a 0.01 M-0.2 M NaCl linear gradient. The subsequent enzymatic condensation of nitrophenyl-pA-U with GDP by polynucleotide phosphorylase in the presence of RNase T, followed similar lines. The final nitrophenyl-pA-U-G was catalytically reduced to aminophenyl-pA-U-G and then bromoacetylated as described for the reduction and bromoacetylation of nitrophenyl-dTp (11). A detailed procedure of the synthesis of Nacylated aminophosphorylated oligonucleotides will be published elsewhere.

Toward the Characterization and Identification of γ‐Secretases Using Transition‐state Analogue Inhibitors

“…To identify the target of these γ‐secretase inhibitors, we have designed derivatives of these peptide analogues that may bind covalently and irreversibly. The N‐terminal Boc group of 1b was replaced with a bromoacetyl group, a functionality susceptible to nucleophilic attack that has been extensively employed as a means of creating affinity labels 32–37. Thus, the difluoro ketone transition‐state mimic should interact with the two active site aspartates of β‐secretase,38 while the bromoacetamide should react with any proximal nucleophilic residues (e.g., Ser, Cys, Thr) to form a stable covalent bond and irreversibly inactivate the enzyme.…”

“…The N-terminal Boc group of 1b was replaced with a bromoacetyl group, a functionality susceptible to nucleophilic attack that has been extensively employed as a means of creating affinity labels. [32][33][34][35][36][37] Thus, the difluoro ketone transition-state mimic should interact with the two active site aspartates of γ-secretase, 38 while the bromoacetamide should react with any proximal nucleophilic residues (e.g., Ser, Cys, Thr) to form a stable covalent bond and irreversibly inactivate the enzyme. As expected, we found that the Boc-containing 1b reversibly inhibits γ-secretase: Upon replacement with inhibitorfree media, Aβ production returned to control levels (FIG.…”

Synthesis of a Chemically Reactive Analog of the Nonsense Codon U-G-A. Its Reaction with Ribosomes of Escherichia coli

“…{4-Bromo-[2][3][4][5][6][7][8][9][10][11][12][13][14] C\acetamidophenyl)phospho} uridylyl-(3'-5')-guanylyl-(3'-5')-adenosine (V) 0.65 μπιοί compound IV in IM phosphate buffer (pH6.0) was mixed with 1.3 μηιοί 2,2'-dibromo-[2-14 C]acetic anhydride (54 Ci/mol) and allowed to react for 10 h in the dark at room temperature. The reaction mixture was subjected to descending preparative paper chromatography in solvent system B (developing time -40 h).…”