An element within the 5' untranslated region of human Hsp70 mRNA which acts as a general enhancer of mRNA translation.
The untranslated regions of mRNAs encoding heat-shock proteins have been reported to contain elements important to the post-transcriptional regulation of these key components of the stress response. In this report we describe an element from the 5'UTR of human Hsp70 mRNA that increases the efficiency of mRNA translation. Cloning of this region upstream of the coding sequence of two different reporter genes (firefly luciferase and chloramphenicol acetyltransferase) increases expression of the reporter under normal cell culture conditions by up to an order of magnitude. This effect was observed in three different promoter contexts (HSP, SV40 and CMV) and in six cell lines. The increase in protein production is not accompanied by any alteration in mRNA levels, suggesting that the element facilitates translation. 5' or 3' truncated sequences are ineffective in enhancing reporter expression, suggesting that the activity arises from the secondary structure of the element, rather than from some smaller defined motif. Computer analysis of this region revealed that it is able to form stable secondary structures (DeltaG approximately -292.6 kJ x mol(-1)). The Hsp70 element does not seem to act as an internal ribosome entry site. Incorporation of the sequence into plasmids used for DNA vaccination produces increased antibody responses, confirming that the sequence is functional in primary cells. These data suggest that the 5'UTR of human Hsp70 mRNA plays an important role in determining Hsp70 expression levels, and that it contains an element of general utility in enhancing recombinant protein expression systems.
Laboratoire GlaxoWellcome, Les Ulis, France.
S
S
Vivinus
S
S
Baulande
M
M
van Zanten
F
F
Campbell
P
P
Topley
J H
JH
Ellis
P
P
Dessen
H
H
Coste
D020121:5' Untranslated Regions / Q000235:genetics*; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000276:immunology; D002352:Carrier Proteins / Q000235:genetics; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D000091342:Endoplasmic Reticulum Chaperone BiP; D016228:Endothelial Growth Factors / Q000235:genetics; D017930:Genes, Reporter; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006360:Heat-Shock Proteins; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008222:Lymphokines / Q000235:genetics; D018832:Molecular Chaperones / Q000235:genetics; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry*; D019444:Vaccines, DNA / Q000276:immunology; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D020121:5' Untranslated Regions; D000952:Antigens, Polyomavirus Transforming; D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D016228:Endothelial Growth Factors; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008222:Lymphokines; D018832:Molecular Chaperones; D012333:RNA, Messenger; D019444:Vaccines, DNA; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase