An unstructured mRNA region and a 5' hairpin represent important elements of the E. coli translation initiation signal determined by using the bacteriophage T7 gene 1 translation start site.
Gene 1 of bacteriophage T7 early region--the RNA polymerase gene--is very actively translated during the infectious cycle of this phage. A 29 base pair fragment of its ribosome binding site containing the initiation triplet, the Shine-Dalgarno sequence (S-D), 10 nucleotides (nt) upstream and 6 nt downstream of these central elements was cloned into a vector to control the expression of the mouse dihydrofolate reductase gene (dhfr). Although all essential parts of this translation initiation region (TIR) should be present, this fragment showed only very low activity. Computer analysis revealed a potentially inhibitory hairpin binding the S-D sequence into its stem base paired to vector-derived upstream sequences. Mutational alterations demonstrated that this hairpin was not responsible for the low activity. However, addition of 21 nt of the T7 gene 1 upstream sequence to the 29 base pair fragment were capable of increasing the translational efficiency by one order of magnitude. Computer analysis of this sequence, including nucleotide shuffling, revealed that it contains a highly unstructured region lacking mRNA secondary structures but with a hairpin at its 5' end, here formed solely by T7 sequences. There was not much difference in activity whether the mRNA included or lacked vector-derived sequences upstream of the hairpin. Such highly unstructured mRNA regions were found in all very efficiently expressed T7 genes without any obvious sequence homologies. The delta G values of these regions were higher, i.e. potential secondary structural elements were fewer, than in TIR of genes from E. coli. This is likely due to the fact that T7 as a lytic phage is relying for successful infection on much stronger signals which a cell cannot afford because of the indispensable balanced equilibria of its interdependent biochemical processes. When the 5' ends of efficient T7 gene mRNA are formed by the action of RNase III they generally start with an unstructured region. Efficiently expressed T7 genes within a polycistronic mRNA, however, always contain a hairpin preceding the structure free sequence. We suggest that the formation of this 5' hairpin is releasing enough energy to keep the unstructured regions free of secondary RNA structures for sufficient time to give ribosomes and factors a good chance for binding to the TIR. In addition, sequences further downstream of the start codon give rise to an additional increase in efficiency of the TIR by almost two orders of magnitude.
Helke
A
A
University of Heidelberg, Germany.
Geisen
R M
RM
Vollmer
M
M
Sprengart
M L
ML
Fuchs
E
E
D017123:Bacteriophage T7 / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D014764:Viral Proteins
D004269:DNA, Bacterial; D004279:DNA, Viral; D012333:RNA, Messenger; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases