DNA AND RNA OLIGOMER SEQUENCES FROM THE 3'-NONCODING REGION OF THE CHICKEN GLUTAMINE-SYNTHETASE GENE FORM INTRAMOLECULAR HAIRPINS

The DNA sequence of the chicken glutamine synthetase gene contains an A T-rich stretch of approximately 1500 base pairs in the 3' noncoding regions of exon 7 [Pu, H., & Young, A. P. (1989) Gene 18, 169-175]. Wi thin this region several palindromic sequences occur that could concei vably form intramolecular structures. One such perfect inverted repeat sequence resides between positions 2605 and 2623. To investigate the hairpin-forming potential for this sequence, optical and calorimetric melting and gel electrophoresis studies have been performed on the fol lowing synthetically prepared DNA and RNA oligomer subsequences: DNA, 5'd-T-T-T-T-T-T-A-A-T-A-A-T-T-A-A-A-A-A-A-3' and RNA, 5'r-U-U-U-U-U-U- A-A-U-A-A-U-U-A-A-A-A-A-A-3' The DNA strand corresponds to the coding strand sequence while the RNA strand represents the transcribed mRNA. Results of melting analysis of these 19-base, partially self-complemen tary strands performed in 115 mM Naf yielded evaluations of their ther modynamic transition parameters. These values are consistent with the melting of unimolecular structures, presumably hairpins. Thermodynamic parameters evaluated by analysis of the optical melting transitions a ssuming a two-state model and measured directly by differential scanni ng calorimetry agreed within experimental error. Therefore, melting be havior of the hairpins is all-or-none like. The DNA hairpin is slightl y more stable than the RNA hairpin with melting enthalpy Delta H-0 = 4 1.2 +/- 3.8 kcal/mol and entropy QSO = 133 +/- 11 cal/K . mol (eu) com pared to Delta H-0 = 32.0 +/- 6.0 kcal/mol and entropy Delta S-0 = 105 /- 20 eu for the RNA. Gel electrophoretic analysis of these oligomers alone and in various mixtures with their DNA and RNA complementary st rands was also performed. Consistent with interpretations of melting r esults, these experiments revealed both strands alone preferentially f orm intramolecular hairpin structures. In mixtures in which their comp lementary strands are in vast molar excess (stoichiometric ratios > 10 :1), the intramolecular structures are converted to intermolecular dup lexes. For the DNA and RNA strands examined, the conversion is not com plete until over a 1000-fold excess of the complementary strand is add ed. Semiquantitative analysis of gel electrophoretograms enabled evalu ations of the relative free energies of the hairpin and duplex states as a function of complementary strand concentration. With the finding that these sequences preferentially form hairpins, potential roles the se structures could play in regulatory activities are considered.