Synthesis and characterization of 5-[(4-Azidophenacyl)thio]uridine 5'-triphosphate, a cleavable photo-cross-linking nucleotide analogue.
Department of Biological Chemistry, California College of Medicine, University of California, Irvine 92717. Biochemistry
(Impact Factor: 3.02).
08/1989; 28(14):5814-20. DOI: 10.1021/bi00440a017
The synthesis, isolation, and characterization of a new photo-cross-linking uridine 5'-triphosphate analogue are described. This nucleotide analogue, 5-[(4-azidophenacyl)thio]uridine 5'-triphosphate (5-APAS-UTP), contains an aryl azide group approximately 10 A from the uridine ring. The azide is photoactivated by irradiation at 300 nm, resulting in covalent attachment of the nucleotide to adjacent molecules. The nucleotide can be desulfurated with Raney nickel to cause molecular cleavage between the base and the aryl azide. Desulfuration yields uridine 5'-triphosphate and p-azidoacetophenone. If the analogue is cross-linked to another molecule, desulfuration leaves the analogue's acetophenone group attached to that molecule. This effectively leaves behind a molecular tag on molecules that interact with the uridine analogue either as monomeric nucleotide or as part of an RNA molecule. This nucleotide analogue can be incorporated into internal positions in RNA by transcription in vitro with Escherichia coli RNA polymerase. It can therefore be used to examine interactions between RNA and other molecules (e.g., proteins or nucleic acids). Because the sulfur atom can be selectively removed, the covalent bonds formed between analogue-containing RNA and other molecules can be cleaved, when desired, to facilitate identification of the cross-linked molecules and RNA nucleotides in the cross-linked complex.
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ABSTRACT: We have used a self-cleaving RNA molecule related to a subsequence of plant viroids (a "hammerhead") to study the length-dependent folding of RNA produced during transcription by Escherichia coli RNA polymerase. Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues (3'-deoxyNTP's or 3'-O-methylNTP's). When the transcript can form the "hammerhead" structure it self-cleaves to give a truncated product. The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to those generated by using a noncleaving transcript or by using [alpha-thio]ATP in place of ATP. We have shown that 15-18 nucleotides (nt) of RNA past the cleavage point must be synthesized before the transcript can self-cleave within a ternary complex, whereas RNA freed from the complex by heating can cleave with only 3 or more nt present beyond the cleavage point. There are sequence-dependent as well as length-dependent effects. The results suggest that 12 +/- 1 nt are sequestered within the ternary complex and are consistent with the presence of a DNA-RNA hybrid within the transcription bubble, as proposed by others. The results indicate that the "hammerhead" structure does not disrupt the hybrid. It appears that the RNA beyond the hybrid is not restrained by interactions with the enzyme, since the last stem of the self-cleaving structure forms as soon as the RNA composing it emerges from the DNA-RNA hybrid. Self-cleaving of the transcript offers a simple structural probe for studying less well-characterized transcription complexes. The relevance of the results to models for transcription termination is discussed.
Biochemistry 09/1990; 29(34):7882-90. DOI:10.1021/bi00486a015 · 3.02 Impact Factor
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ABSTRACT: We have isolated Escherichia coli transcription complexes, paused in the presence and absence of Nus A, which contain RNA substituted at every UMP residue with a photocrosslinking nucleotide analog. The pause site is immediately downstream from an RNA stem-loop structure, and although pausing occurs in the absence of Nus A, it is substantially enhanced in the presence of Nus A. We have analyzed the secondary structure of this RNA and show that the analog does not interfere with the formation of the normal stem-loop structures. Additionally, the analog substrate does not alter transcriptional pausing, in the presence or absence of Nus A, indicating that Nus A recognition of the transcription complex is not affected by the presence of the crosslinking groups in the RNA. Ribonuclease digestion of the RNA in paused complexes identifies two accessible regions, two nucleotides in the loop and one near the base of the upstream side of the stem-loop. Cleavage at one loop nucleotide is enhanced by Nus A, while the nucleotide near the base of the stem-loop is partially protected. Upon irradiation of the transcription complex, Nus A is not photoaffinity labeled by the RNA, even at a high molar ratio to RNA polymerase (250: 1). Both the β and β′ subunits are labeled, however, indicating that the putative stem-loop binding domain on the core polymerase involves both subunits. Because the nucleotide protected from ribonuclease by Nus A is very near two analogs, yet Nus A is not crosslinked to the RNA, it is unlikely that Nus A could be protecting this position through direct contact. Furthermore, analog is substituted at positions in both the loop and at several positions in the stem, and again, no crosslinking to Nus A is observed. We conclude that enhancement of pausing by Nus A probably does not require direct interaction with the bases in the RNA stem-loop.
Journal of Molecular Biology 06/1991; 219(1-219):11-25. DOI:10.1016/0022-2836(91)90853-X · 4.33 Impact Factor
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ABSTRACT: We have used photocrosslinking to analyze the contacts between the 3′ end of the RNA and Escherichia coli RNA polymerase during the early steps of RNA synthesis using the nucleotide analog 8-azido-ATP (8-N3-ATP). The crosslinking group on 8-N3-ATP contacts the β, β' and σ subunits when the analog is bound to the holoenzyme. We show here that 8-N3-ATP is a substrate for E. coli RNA polymerase and acts as an RNA chain terminator when incorporated into the 3′ end of nascent RNA. 8-N3-AMP was incorporated uniquely at the 3′ end of tri-, tetra- and pentanucleotides synthesized from a poly[d(A-T)] template and at the 3′ end of pentanucleotides from two promoters (λ PSpr′ and E. coli rrnB P1). The oligonucleotides were covalently attached to the RNA polymerase by irradiation of transcription complexes with ultraviolet light. All RNAs labeled the β and β' subunits, but σ was contacted only by the trinucleotide and tetranucleotide on poly[d(A-T)]. Sigma is still present in transcription complexes containing the pentanucleotide on poly[d(A-T)], despite the lack of labeling. Neither pentanucleotide from the authentic promoters contacted σ. We conclude that as holoenzyme moves downstream, either two separate conformational changes occur, after synthesis of the trinucleotide and tetranucleotide, which result in movement of σ away from the nucleotide binding site or, alternatively, σ remains fixed relative to the DNA while the domain on core polymerase forming the nucleotide binding site moves downstream.
Journal of Molecular Biology 08/1991; 220(2-220):227-239. DOI:10.1016/0022-2836(91)90009-U · 4.33 Impact Factor
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