Stephen S. Hixson

Photochemical labeling of yeast alcohol dehydrogenase with an azide analog of NAD

Abstract— 3‐Azidopyridine adenine dinucleotide, an azide analog of NAD+, has been synthesized as a potentially general photochemical labeling reagent for the active sites of NAD‐dependent dehydrogenases. The analog is a competitive inhibitor of NAD reduction by yeast alcohol dehydrogenase (YADH). Upon photolysis of the 3H‐analog in the presence of YADH, 7% of the label becomes covalently bound. The results are discussed in terms of desired properties of a photochemical labeling reagent.

[82] Photoaffinity-probe-modified tRNA for the analysis of ribosomal binding sites

Publisher Summary This chapter explores the Photoaffinity-Probe-Modified tRNA for the Analysis of Ribosomal Binding Sites, based on the principle: The use of tRNA suitably modified with a chemically reactive group in order to probe the nature of the tRNA binding site in macromolecular complexes such as ribosomes, aminoacyl-tRNA (AA-tRNA) synthetases, and elongation factors is a logical extension of the use of chemically reactive substrate analogs to study the interaction of these small-molecular-weight ligands with proteins. In the same way that changing the location of the chemically reactive group on small molecule substrates alters the probability for covalent linking and thus provides a crude topographical analysis of the binding site, varying the site of the affinity probe on the tRNA molecule can also be expected to affect the extent of covalent binding and thus provide some insight into the contact areas between a tRNA molecule and its binding site. The chapter describes the probes which are photoaffinity probes of the aromatic azide class. It explains the use of p-Azidophenacyl bromide (APA-Br) and its analog, the p-azidophenaeyl ester of bromoacetic acid (APAA-Br) to modify the 4 S residue, while the N-hydroxysuccinimide ester of N-(4-azido-2-nitrophenyl)glycine (NAG-NOS) was used to derivatize the nbt 2 U residue which has a free α-NH 2 group.

Recombinant photoreactive tRNA molecules as probes for cross-linking studies.

Photoreactive tRNA derivatives have been used extensively for investigating the interaction of tRNA molecules with their ligands and substrates. Recombinant RNA technology facilitates the construction of such tRNA probes through site-specific incorporation of photoreactive nucleosides. The general strategy involves preparation of suitable tRNA fragments and their ligation either to a photoreactive nucleotide or to each other. tRNA fragments can be prepared by site-specific cleavage of native tRNAs, or synthesized by enzymatic and chemical means. A number of photoreactive nucleosides suitable for incorporation into tRNA are presently available. Joining of tRNA fragments is accomplished either by RNA ligase or by DNA ligase in the presence of a DNA splint. The application of this methodology to the study of tRNA binding sites on the ribosome is discussed, and a model of the tRNA-ribosome complex is presented.

Preparation of 2-azidoadenosine 3′,5′-[5′-32P]bisphosphate for incorporation into transfer RNA Photoaffinity labeling of Escherichia coli ribosomes

2‐Azidoadenosine was synthesized from 2‐chloroadenosine by sequential reaction with hydrazine and nitrous acid and then bisphosphorylated with pyrophosphoryl chloride to form 2‐azidoadenosine 3′,5′‐bisphosphate. The bisphosphate was labeled in the 5′‐position using the exchange reaction catalyzed by T4 polynucleotide kinase in the presence of [γ‐32P]ATP. Polynucleotide kinase from a T4 mutant which lacks 3′‐phosphatase activity (ATP:5′‐dephosphopolynucleotide 5′‐phosphotransferase, EC 2.7.1.78) was required to facilitate this reaction. 2‐Azidoadenosine 3′,5′‐[5′‐32P]bisphosphate can serve as an efficient donor in the T4 RNA ligase reaction and can replace the 3′‐terminal adenosine of yeast tRNAPhe with little effect on the amino acid acceptor activity of the tRNA. In addition, we show that the modified tRNAPhe derivative can be photochemically cross‐linked to the Escherichia coli ribosome.

A Model of the tRNA Binding Sites on the Escherichia Coli Ribosome

In the course of the elongation cycle of translation, three different functional forms of tRNA bind to mRNA-programmed ribosomes. Two of them, aminoacyl-tRNA and peptidyl-tRNA, participate in the formation of the peptide bond while accommodated in the A (acceptor) and P (peptidyl) sites. A third form, deacylated tRNA, a product of the transpeptidation reaction, is transferred from the P site to the E (exit) site during translocation before it leaves the ribosome. Since binding of tRNA to the ribosome is essential for protein synthesis, numerous studies have been designed to delineate the topography of the ribosomal A, P and E sites (for review see Cooperman, 1980; Ofengand et al., 1986; Cooperman, 1987).

Photochemical cross-linking of the anticodon loop of yeast tRNAPhe to 30S-subunit protein S7 at the ribosomal A and P sites

Yeast tRNA(Phe), containing the photoreactive nucleoside 2-azidoadenosine at position 37 within the anticodon loop, has been cross-linked to the aminoacyl-tRNA (A) and peptidyl-tRNA (P) binding sites of the Escherichia coli ribosome. The 30S subunit was exclusively labeled in each case, and cross-linking occurred to both protein and 16S rRNA. Electrophoretic and immunological analyses demonstrated that S7 was the only 30S-subunit protein covalently attached to the tRNA. However, digestion of the A and P site-labeled S7 with trypsin revealed a unique pattern of cross-linked peptide(s) at each site. Thus, while the anticodon loop of tRNA is in close proximity to protein S7 at both the A and P sites, it neighbors a different portion of the protein molecule in each. The placement of the aminoacyl- and peptidyl-tRNA binding sites is discussed in relationship to recent models of the 30S ribosomal subunit.

Transit of tRNA through the Escherichia coli ribosome: cross-linking of the 3 ' end of tRNA to ribosomal proteins at the P and E sites

Photoreactive derivatives of yeast tRNAPhe containing 2‐azidoadenosine at their 3′ termini were used to trace the movement of tRNA across the 50S subunit during its transit from the P site to the E site of the 70S ribosome. When bound to the P site of poly(U)‐programmed ribosomes, deacylated tRNAPhe, Phe‐tRNAPhe and N‐acetyl‐Phe‐tRNAPhe probes labeled protein L27 and two main sites within domain V of the 23S RNA. In contrast, deacylated tRNAPhe bound to the E site in the presence of poly(U) labeled protein L33 and a single site within domain V of the 23S rRNA. In the absence of poly(U), the deacylated tRNAPhe probe also labeled protein L1. Cross‐linking experiments with vacant 70S ribosomes revealed that deacylated tRNA enters the P site through the E site, progressively labeling proteins L1, L33 and, finally, L27. In the course of this process, tRNA passes through the intermediate P/E binding state. These findings suggest that the transit of tRNA from the P site to the E site involves the same interactions, but in reverse order. Moreover, our results indicate that the final release of deacylated tRNA from the ribosome is mediated by the F site, for which protein L1 serves as a marker. The results also show that the precise placement of the acceptor end of tRNA on the 50S subunit at the P and E sites is influenced in subtle ways both by the presence of aminoacyl or peptidyl moieties and, more surprisingly, by the environment of the anticodon on the 30S subunit.

ELECTRON-TRANSFER MEDIATED ADDITION OF METHANOL TO BENZOBICYCLO[3.1.0]HEX-2-ENE

Abstract The photochemically-prepared cation radical of benzobicyclo[3.1.0]hex-2-ene undergoes nucleophilic addition of methanol at C 5 and C 6 in a ratio of approximately 5:3.

Bifunctional Aryl Azides as Probes of the Active Sites of Enzymes

A series of five inhibitors was used in a continuing investigation of the use of aryl azides to label the active site regions of isolated enzymes in solution. In each case, photolysis of the enzyme-inhibitor system while the inhibitor was at the active site should have produced a nitrene from the aryl azide portion of the inhibitor. This nitrene was expected to insert into a nearby portion of the protein chain. Identification of the insertion sites provides information about the three-dimensional structure of the active site of the enzyme in solution. The initial three inhibitor-enzyme systems studied provided useful general information about the possibilities and problems involved in this general method, while the last two systems are being pursued to the point of identifying the insertion sites.

Arylcyclopropane photochemistry. Substituent effects on the photochemical conversion of 1,1-diarylcyclopropanes to 1,1-diarylpropenes and 1-arylindanes.

Abstract The rate of photochemical rearrangement of 1,1-diarylcyclopropanes to 1,1-diarylpropenes and 1-arylindanes is enhanced by electron-withdrawing groups on the aromatic rings and diminished by electron-donating groups.