Prevention of chain cleavage in the chemical synthesis of 2′-silylated oligoribonucleotides

150

149

Expression of the structural proteins of human immunodeficiency virus type 1 requires the direct interaction of multiple copies of the viral Rev protein with its highly structured RNA target sequence, the Rev response element (RRE). Nucleotides critical for Rev monomer binding have been mapped by chemical interference to a single site flanking the base of an RNA helix (stem HB) located within the 234-nucleotide RRE. Binding of additional Rev molecules to an RRE probe did not require any RNA primary sequence information detectable by modification interference beyond that required for binding of a single Rev protein molecule. A synthetic 29-nucleotide RNA molecule designed to incorporate nucleotides identified as critical for Rev binding retained the ability to bind Rev specifically and, therefore, represents a minimal Rev-binding site. We propose that Rev binding to the RRE initiates with the direct interaction of a Rev monomer with a high-affinity binding site located at the base of the IIB stem of the RRE. The subsequent formation of Rev multimers on the RRE appears, in contrast, primarily driven by specific protein-protein interactions.The Rev gene product of human immunodeficiency virus type 1 (HIV-1) is an RNA-sequence-specific nuclear regulatory protein essential for HIV-1 replication in culture (1-6). The target sequence for Rev, the rev response element (RRE), was initially defined as a complex 234-nucleotide (nt) RNA stem-loop structure located within the viral env gene (7). At least two, and possibly as many as eight, Rev molecules have been shown to bind specifically to the full-length [8][9][10]. Footprinting experiments suggest that these Rev protein molecules occupy discrete sites on the RRE (10). Although we and others have argued that the formation of Rev multimers on the RRE reflects the sequential binding of multiple Rev monomers (8, 9), it has also been suggested that the formation of multimers may be a prerequisite for Rev binding (11).Mutational analysis of the 116-amino acid Rev protein has shown that an arginine-rich stretch of amino acids is critical for sequence-specific binding to the RRE, whereas flanking sequences are important for the formation of Rev multimers on the RRE (8,11,12). It has therefore been proposed that this Rev "multimerization" is mediated, at least in part, by protein-protein interactions (8,9,11,12). Rev mutants that have lost the ability to form multimers on the RRE in vitro display a recessive negative phenotype in vivo (8,11).Mutational analysis of the RRE has defined a 66-nt subdomain, termed stem-loop II (SLII) (Fig. 1A), that is both necessary and sufficient for high-affinity Rev binding in vitro (4,5) and that displays at least partial RRE function in vivo (16). This RRE subdomain can bind up to three Rev molecules in vitro (9, 10). More recently, a 40-nt sequence contained within SLII has been proposed as a minimal RNA substrate based on its ability to bind a single Rev monomer (9). Here, we identify several noncontiguous nucleotides located within S...

Section: Methodsmentioning

confidence: 99%

Identification of a high-affinity RNA-binding site for the human immunodeficiency virus type 1 Rev protein.

Tiley

Malim

Tewary

et al. 1992

150

149

“…Oligoribonucleotides were synthesized by standard methods (16)(17)(18), and one hairpin was extended by adding pUp with the T4 RNA ligase reaction (19,20). Synthetic details will be provided elsewhere.…”

mentioning

confidence: 99%

Coaxial stacking of helixes enhances binding of oligoribonucleotides and improves predictions of RNA folding.

Walter

Turner

Kim

et al. 1994

450

327

An RNA model system consisting of an oligomer binding to a 4-nt overhang at the 5' end ofa hairpin stem provides thermodynamic parameters for helix-helix interfaces. In a sequence-dependent manner, oligomers bind up to 1000-fold more tightly adjacent to the hairpin stem than predicted for binding to a free tetramer at 370C. For reaction (19, 20). Synthetic details will be provided elsewhere.Measurement of Thermodynamic Parameters. The buffer for melting experiments was 1 M NaCl/10 mM sodium cacodylate/0.5 mM EDTA. Absorbance versus temperature curves were measured at 280 nm with a heating rate of 10C/min. These curves were analyzed by fitting to a two-state model with sloping base lines through use of a nonlinear least-squares program (21).Predictions of Secondary Structure. Sets of 20 secondary structures were generated with the program of Zuker (12), kcal/mol, and internal loops of three were given a AG'7 for loop closure of 5.1 + 0.3 = 5.4 kcal/mol (23). (iii) Stacking and pairing of terminal mismatches were included in calculating free energies of internal loops in the following manner. Single mismatches were given AG7 = 0.8 kcal/mol. For larger internal loops, terminal GA, UU, and other mismatches adjacent to COG pairs were given favorable free energy increments of -2.7, -2.5, and -1.5 kcal/mol, respectively (24). These mismatches adjacent to A-U pairs were given AG' values of -2. 9218The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

“…Oligonucleotides were synthesized chemically on an Applied Biosystems model 380B DNA synthesizer as described (15,16) (12) accounted for <2% of the maximal observed rate. This background rate was subtracted from each data point, although this had no effect on the fit.…”

mentioning

confidence: 99%

Guanosine binding to the Tetrahymena ribozyme: thermodynamic coupling with oligonucleotide binding.

McConnell

Cech

Herschlag

1993

184

The L-21 Sca I ribozyme derived from the group I intron of Tetrahymena thrmophila pre-rRNA catalyzes an endonucease reaction analogous to the first step of selfspicing. Guanosine (G) The 413-base intron of Tetrahymena thermophila nuclear pre-rRNA splices in the absence of protein (1, 2). In the first of two transphosphoesterification steps, exogenous guanosine (G) or one of its 5' phosphorylated forms binds to the intron and attacks at the 5' splice site. The products of the first step are the 5' exon ending with a 3'-hydroxyl group and the intron-3' exon species with the attacking G covalently linked at the 5' end. These products then undergo the second step of splicing, a reaction that is chemically equivalent to the reverse of the first step with the 3'-terminal guanosine of the intron at position 414 (G414) substituting for the exogenous G. This reaction produces ligated exons and an excised intron. The L-21 Sca I RNA is a shortened form of the intron that catalyzes an intermolecular reaction analogous to the first step of splicing (Fig. 1). This form of the intron cleaves an RNA substrate of specific sequence with multiple turnover and, thus, can be considered an RNA enzyme, or "ribozyme." Kinetic and thermodynamic characterization of this ribozyme has led to a new level of mechanistic understanding of the intron chemistry and biology (10).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.In previous work, however, assumptions were made in determining the ribozyme's affinity for G (11)(12)(13) with 2-3 volumes of stop buffer containing 80% formamide, 50 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol, and 2 mM Tris borate, pH 7.5. Reaction products were separated on 20% polyacrylamide/7 M urea gels. When reactions were followed for more than 2 hr, the mixtures were kept submerged and/or were centrifuged periodically to prevent concentration of the sample by evaporation. The fraction of substrate remaining relative to total substrate and Abbreviations: pG, guanosine 5'-monophosphate; E, the Tetrahymena ribozyme; S, an oligonucleotide substrate whose identity depends on the experiment; kc, the rate constant for the chemical step, which is equal to kt for the single-turnover reactions herein.