Affinity selection-amplification from randomized ribooligonucleotide pools.
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Amino Acids↗
Biomedical subjects
S Zinnen
Publications and source records attributed to S Zinnen.
An RNA pocket for the planar aromatic side chains of phenylalanine and tryptophane.
This is a preliminary report of RNA's with affinity for the aromatic side chains of the amino acids phenylalanine and tryptophane that have been isolated utilizing in vitro selection to phenylalanine-affinity matrix. Cloning and sequencing of a binding pool identified a frequently occurring 6-9 base motif (-CUCGUGU-) common to most of the RNA's; other potential binding motifs were revealed but occurred less frequently. Four of five clones analyzed bind free phenylalanine and tryptophane with dissociation constants in the low millimolar range, the fifth binds resin only. Isolate F7, the most common sequence in the binding pool, demonstrated moderate specificity for aromatic rings, no stereoselectivity was observed. Preliminary Pb cutting structural analysis agrees with the most stable predicted secondary structure. A striking similarity between the sequence and predicted structure of F7 and the bridged-biphenylisomerase RNA (J.R. Prudent, T. Uno, and P.G. Schultz, Science 264,1924 (1994)) suggests a 19 base loop contains the important aromatic binding elements.
Amino Acid Sequence↗
Misincorporation and mispaired primer extension by human immunodeficiency virus reverse transcriptase.
Pre-steady-state methods were used to study the fidelity of human immunodeficiency virus reverse transcriptase. Fidelity of DNA-directed DNA synthesis can be attributed to a 1-2 order of magnitude reduction in affinity for noncomplementary dNTPs, and a 1-4 order of magnitude reduction in the rate of the conformational change that limits the rate of nucleotide addition. Affinities of reverse transcriptase for paired or mispaired primer termini are similar. Discrimination against a mispaired primer is due to reduction in affinity for the next dNTP and reduction in rate of extension. Extension of mispaired termini proceeds 20-700-fold faster than the rate of dissociation of reverse transcriptase from the primer-template and is 2-3 orders of magnitude more frequent than nucleotide misincorporation. The rate-limiting step for extension of a mispaired terminus occurs at the conformational change or chemical step, depending on the nature of the mispair. Presence of a mismatch at the 3' penultimate position reduces pyrophosphorolysis of the primer by a factor of 10(3), indicating that mispairs 5' to the site of chemistry can also affect catalysis.
Base Composition↗
Kinetic mechanism of the DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase.
The kinetic pathway of DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase (HIV RT) as determined by pre-steady-state methods using a defined primer/template is as follows, [formula: see text] where E is RT, Dn,n+1 is primer/template, dNTP is deoxyribonucleoside triphosphate, and PPi is pyrophosphate. The rate-determining step for enzyme turnover in single nucleotide addition is the dissociation of enzyme from DNA (k6 = 0.11 s-1). The observation of an E'.DNA.dNTP intermediate by pulse-chase analysis and the absence of a phosphorothioate elemental effect identified the rate-limiting step for nucleotide addition as a conformational change of the E.DNA.dNTP complex (k3 = 83 s-1) prior to the chemical step. Biphasic kinetics of single-turnover pyrophosphorolysis suggested that this conformational change (k-3 = 0.3 s-1) is also rate-limiting for the reverse reaction. The equilibrium constant for the chemical step (K4) is 3.8, in slight favor of the forward reaction. The large equilibrium constant (K3 = 280) for the conformational change effectively renders nucleotide addition kinetically irreversible. The dissociation constant for primer/template is 26 nM, and the association rate of enzyme and DNA (k1) is 2.3 x 10(6) M-1 s-1. Equilibrium dissociation constants for dTTP and PPi are 18 microM and 7.2 mM, respectively. Mg2+ enhances productive interaction of RT with DNA as judged by a 50% increase in burst amplitude in the single nucleotide addition reaction and by an 8-fold decrease in KD for the RT.DNA complex as determined by gel mobility shift assay. Secondary interactions of the RT.DNA complex with free DNA were observed in the absence of Mg2+.
Base Sequence↗