Determinants of ribose specificity in RNA polymerization: Effects of Mn2+ and deoxynucleoside monophosphate incorporation into transcripts

Ying Huang, Amber Beaudry, Jim McSwiggen, Rui Sousa

Research output: Contribution to journalArticle

50 Scopus citations

Abstract

The catalytic specificity of T7 RNA polymerase (RNAP) for ribonucleoside triphosphates vs deoxynucleoside triphosphates {(k(cat)/K(m))(rNTP)/(k(cat)/K(m))(dNTP)} during transcript elongation is ~80. Mutation of tyrosine 639 to phenylalanine reduces specificity by a factor of ~20 and largely eliminates the K(m) difference between rNTPs and dNTPs. The remaining specificity factor of ~4 is k(cat)-mediated and is nearly eliminated if Mn2+ is substituted for Mg2+ in the reaction. Mn2+ substitution does not significantly affect the K(m) difference between rNTPs and dNTPs. Mn2+ substitution also enhances the activity of poorly active mutant enzymes carrying nonconservative substitutions in the active site, and its effects are generally consistent with the Mn2+-catalyzed reaction being less restrictive in its requirements for alignment of the reactive groups. In addition to discrimination occurring at the level of nucleoside monophosphate (NMP) incorporation, it is also found that transcripts containing deoxynucleoside monophosphates (dNMPs) are more poorly extended than transcripts of canonical structure, though a severe barrier to transcript extension is seen only when the 3' region of the transcript is heavily substituted with dNMPs. The barrier to extension of transcripts heavily substituted with dNMPs is reduced for sequences known to be amenable to forming A-like helices and is larger for sequences that resist transformation from B-form DNA·DNA structures. The barrier to extension of dNMP-substituted transcripts is also reduced by solution conditions known to destabilize B- form DNA and to stabilize A-form structures. These observations imply a requirement for a non-B-form, possibly A-like, conformation in the transcript·template hybrid that is disrupted when the transcript is of predominantly deoxyribose structure.

Original languageEnglish (US)
Pages (from-to)13718-13728
Number of pages11
JournalBiochemistry
Volume36
Issue number44
DOIs
StatePublished - Nov 4 1997

ASJC Scopus subject areas

  • Biochemistry

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