Post-transcriptional control of bacteriophage T4 gene 25 expression: mRNA secondary structure that enhances translational initiation

Secondary structure of the mRNA in the translational initiation region is a n important determinant of translation efficiency. However, the secondary s tructures that enhance or facilitate translation initiation are rare. We ha ve previously proposed that such structure may exist in the case of bacteri ophage T4 gene 25 translational initiation region, which contains three pot ential Shine-Dalgarno sequences (SD1, SD2, and SD3) with a spacing of 8, 17 , and 27 nucleotides from the initiation codon of this gene, respectively. We now present results that clearly demonstrate the existence of a hairpin structure that includes SD1 and SD2 sequences and brings the SD3, the most typical of these Shine-Dalgarno sequences, to a favourable spacing with the initiation codon of gene 25. Using a phage T7 expression system, we show that mutations that prevent the formation of hairpin structure or eliminate the SD3 sequence result in a d ecreased level of gp25 synthesis. Double mutation in basepair V restores th e level of gene 25 expression that was decreased by either of the two mutat ions (C-to-G and G-to-C) alone, as predicted by an effect attributable to m RNA secondary structure. We introduced the mutations into the bacteriophage T4 by plasmid-phage recombination. Changes in the plaque and burst sizes o f T4 mutants, carrying single and double mutations in the translational ini tiation region of gene 25, strongly suggest that the predicted mRNA seconda ry structure controls (enhances) the level of gene 25 expression in vivo. H ybridization of total cellular RNA with a gene 25 specific probe indicated that secondary structure or mutations in the translational initiation regio n do not notably affect the 25 mRNA stability. Immunoblot analysis of gp25 in Escherichia coli cells infected by T4 mutants showed that mRNA secondary structure increases the level of gp25 synthesis by three- to fourfold. Sin ce the secondary structure increases the level of gp25 synthesis and does n ot affect mRNA stability, we conclude that this structure enhances translat ion initiation. We discuss some features of two secondary structures in the translational initiation regions of T4 genes 25 and 38. (C) 1999 Academic Press.