Mg2+ binding and structural stability of mature and in vitro synthesized unmodified Escherichia coli tRNAPhe.

Abstract:

:Mature tRNAPhe from Escherichia coli and the transcript of its gene lacking modified nucleotides were compared by a variety of physical techniques. Melting experiments revealed that at a low Mg2+level the transcript was partially denatured, while the mature tRNA possessed intact tertiary interactions. Mg2+binding to both tRNAs was studied by CD and UV techniques as well as by using the Mg2+-sensitive fluorescence indicator, 8-hydroxyquinoline 5-sulfonic acid. Both tRNA forms exhibited a single strong Mg2+-binding site, its dissociation constant was 10-fold higher for the transcript. Conformational changes in response to Mg2+ addition measured by CD and UV spectrometry revealed no difference for the estimated binding cooperativity and strong differences for affinities of Mg2+-binding sites for the two tRNA forms. Conformational transitions in mature and in in vitro synthesized tRNA required the binding of two Mg2+ ions per molecule and therefore should be associated not only with a single strong binding site. The Mg2+ dependence of Stokes radii measured by gel-filtration revealed insignificant differences between the overall sizes of the two tRNA forms at physiological Mg2+ levels (>1 mM). Taken together, these results suggest that modified nucleotides stabilize tertiary interactions and increase the structure stability without affecting the mechanism of Mg2+binding and overall folding of the tRNA molecule. This conclusion is supported by the known biological activity of the E. coli tRNAPhe gene transcript.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Serebrov V,Vassilenko K,Kholod N,Gross HJ,Kisselev L

doi

10.1093/nar/26.11.2723

subject

Has Abstract

pub_date

1998-06-01 00:00:00

pages

2723-8

issue

11

eissn

0305-1048

issn

1362-4962

pii

gkb464

journal_volume

26

pub_type

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