Small, synthetic, GC-rich mRNA stem-loop modules 5′ proximal to the AUG start-codon predictably tune gene expression in yeast

Table 4 Effects of modifying the core Sfi I stem-loop sequence (GGCCGCTCGGGCC; modifying the size of the stem and the loop) at position −4 to the ATG start codon on the expression of Cdr1p

construct #	5′ UTR⁺	stem-loop type	MIC_FLC⁺⁺(mg/l)	-∆G⁺⁺⁺(kcal/mol)
		loop +
6	TCCGCTCGAGGCC AA GCTCG A GGCCT AAAATG	8	100	8.6
7	TCCGCTCGTTCGAAAGGCC AA GGCCT AAAATG	2	100	6.6
8	TCCGCTCGTTCGAAAGAGGCCGGCCT AAAATG	0	200	2.8
1	TCCGCTCGTTCAGGCC GCTCG GGCCT AAAATG	5	50	8.6
		stem +
5	TCCGCTCGTTCGATT CCGCTCGGG CCAAAATG	−2	200	−0.2
4	TCCGCTCGTTCGAGGCCGCTCGGGC G AAAATG	−1	100	4.4
9	TCCGCTCGAAAAGGCCGCTCGGGCC AAAAATG	-	25	7.9
2	TCCGCTCGTTCC GGCCGCTCGGGCC G AAAATG	C	12.5	9.8
11	TCCGCTCGAAAC GGCCGCTCGGGCC G AAAATG	C	12.5	10.0
14	TCCGCTAAAGC GGCCGCTCGGGCC GC AAAATG	GC	3.2	13.7
15	TCCGAAACGC GGCCGCTCGGGCC GCG AAAATG	CGC	1.6	15.8
1*	TCCGCTCGTTCA GGCCGCTCGGGCC T AAAATG	A	50	8.6
10*	TCCGCTCGAAAT GGCCGCTCGGGCC A AAAATG	T	6.3	8.7
13*	TCCGCTAAATA GGCCGCTCGGGCC TA AAAATG	TA	25	10.0
18*	TCCGAAATTA GGCCGCTCGGGCC TAA AAAATG	TTA	12.5	10.9
12*	TCCGCTAAAGA GGCCGCTCGGGCC TC AAAATG	GA	6.3	12.4
17*	TCCGAAATGA GGCCGCTCGGGCC TCA AAAATG	TGA	3.2	13.2
16*	TCCGAAACGA GGCCGCTCGGGCC TCG AAAATG	CGA	6.3	14.5

⁺ All stem-loop sequences are underlined. Loop nucleotides of the Sfi I stem-loop constructs with different size loops (top) are highlighted in bold type and nucleotide changes made to modify the size of the stem (bottom) of the core Sfi I stem-loop are also highlighted in bold type letters. Numbers at the top panel indicate the size of the loop; a – sign followed by a number indicates by how many nucleotide pairs the core Sfi I stem-loop stem was reduced and letters indicate the nucleotide sequence of the left arm of the nucleotide pairs with which the core Sfi I stem-loop had been increased.
⁺⁺ MIC_FLC values represent the MIC_FLC values of three independent transformants and they were practically identical for all constructs tested.
⁺⁺⁺ ∆G values calculated for individual Sfi I stem-loops (underlined sequences).
* Sfi I stem-loop constructs of different size stems containing mixed AU- and GC-pairs.

ISSN: 1475-2859