DNA and RNA Oligomer Sequences from the 3‘ Noncoding Region of the Chicken Glutamine Synthetase Gene Form Intramolecular Hairpins

• Published in: Biochemistry (Easton) 1996-12, Vol.35 (48), p.15364-15372
• Main Authors: Riccelli, Peter V, Hilario, Jovencio, Gallo, Frank J, Young, Anthony P, Benight, Albert S
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9615203

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]. Within this region several palindromic sequences occur that could conceivably 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 following 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-complementary strands performed in 115 mM Na+ yielded evaluations of their thermodynamic transition parameters. These values are consistent with the melting of unimolecular structures, presumably hairpins. Thermodynamic parameters evaluated by analysis of the optical melting transitions assuming a two-state model and measured directly by differential scanning calorimetry agreed within experimental error. Therefore, melting behavior of the hairpins is all-or-none like. The DNA hairpin is slightly more stable than the RNA hairpin with melting enthalpy ΔH 0 = 41.2 ± 3.8 kcal/mol and entropy ΔS 0 = 133 ± 11 cal/K·mol (eu) compared to ΔH 0 = 32.0 ± 6.0 kcal/mol and entropy ΔS 0 = 105 ± 20 eu for the RNA. Gel electrophoretic analysis of these oligomers alone and in various mixtures with their DNA and RNA complementary strands was also performed. Consistent with interpretations of melting results, these experiments revealed both strands alone preferentially form intramolecular hairpin structures. In mixtures in which their complementary strands are in vast molar excess (stoichiometric ratios > 10:1), the intramolecular structures are converted to intermolecular duplexes. For the DNA and RNA strands examined, the conversion is not complete until over a 1000-fold excess of the complementary strand is added. Semiquantitative analysis of gel electrophoretograms enabled evaluations 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