title: Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.
abstract: BACKGROUND
  A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5' UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC).
  
  
  RESULTS
  Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5' UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = -15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = -4.4 kcal/mol) inhibited Cdr1p expression by ~50%.
  
  
  CONCLUSIONS
  We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression.
authors.affiliation: Sir John Walsh Research Institute, University of Otago, Dunedin 9054, New Zealand. erwin.lamping@otago.ac.nz.
authors.forename: Erwin
authors.initials: E
authors.lastname: Lamping
authors.forename: Masakazu
authors.initials: M
authors.lastname: Niimi
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cannon
mesh_terms: D020121:5' Untranslated Regions; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D020029:Base Pairing; D002176:Candida albicans / Q000378:metabolism; D003001:Cloning, Molecular; D018387:Codon, Initiator; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D020121:5' Untranslated Regions; D018528:ATP-Binding Cassette Transporters; D018387:Codon, Initiator; D005656:Fungal Proteins; C092438:PDR5 protein, S cerevisiae; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins
references.citation: Gene. 2005 Nov 21;361:57-66
references.pmid: 16185821
references.citation: FEMS Yeast Res. 2012 Mar;12(2):144-70
references.pmid: 22136110
references.citation: Mol Cell Biol. 2010 Sep;30(18):4415-34
references.pmid: 20584985
references.citation: Science. 2008 Feb 29;319(5867):1215-20
references.pmid: 18218864
references.citation: Mol Microbiol. 2011 Oct;82(2):416-33
references.pmid: 21895791
references.citation: EMBO J. 2011 Jan 5;30(1):115-29
references.pmid: 21113134
references.citation: FEMS Yeast Res. 2012 Mar;12(2):228-48
references.pmid: 22188344
references.citation: Mol Microbiol. 2006 Oct;62(1):170-86
references.pmid: 16942600
references.citation: J Biol Chem. 1993 Dec 15;268(35):26522-30
references.pmid: 8253781
references.citation: Mol Microbiol. 2012 Aug;85(4):747-67
references.pmid: 22788839
references.citation: Eukaryot Cell. 2007 Jul;6(7):1150-65
references.pmid: 17513564
references.citation: Nucleic Acids Res. 2009 Feb;37(2):e16
references.pmid: 19074487
references.citation: Methods Enzymol. 1999;306:145-53
references.pmid: 10432452
references.citation: Antimicrob Agents Chemother. 2001 Dec;45(12):3366-74
references.pmid: 11709310
references.citation: Jpn J Infect Dis. 2005 Feb;58(1):1-7
references.pmid: 15728981
references.citation: Anal Biochem. 2009 Nov 1;394(1):87-91
references.pmid: 19577533
references.citation: Genome Biol. 2002;3(3):REVIEWS0004
references.pmid: 11897027
references.citation: PLoS Comput Biol. 2005 Dec;1(7):e72
references.pmid: 16355254
references.citation: Mol Cell Biol. 1988 Apr;8(4):1591-601
references.pmid: 2837649
references.citation: Nat Chem Biol. 2008 Sep;4(9):564-73
references.pmid: 18690217
references.citation: Nucleic Acids Res. 1994 Dec 25;22(25):5767-8
references.pmid: 7838736
references.citation: J Biol Chem. 1993 Mar 25;268(9):6453-62
references.pmid: 8454618
references.citation: J Biol Chem. 1994 Jul 15;269(28):18630-7
references.pmid: 8034611
references.citation: Nucleic Acids Res. 2011 Aug;39(14):e92
references.pmid: 21586584
references.citation: Antimicrob Agents Chemother. 2008 Nov;52(11):3851-62
references.pmid: 18710914
references.citation: Cell. 2009 Feb 20;136(4):731-45
references.pmid: 19239892
references.citation: Proc Natl Acad Sci U S A. 2005 Sep 6;102(36):12678-83
references.pmid: 16123130
references.citation: Antimicrob Agents Chemother. 2004 Apr;48(4):1256-71
references.pmid: 15047528
references.citation: Microbiol Mol Biol Rev. 2011 Sep;75(3):434-67, first page of table of contents
references.pmid: 21885680
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3406-15
references.pmid: 12824337
references.citation: Biotechnol J. 2010 Feb;5(2):147-62
references.pmid: 20084640
references.citation: Cell Mol Life Sci. 2008 Jun;65(11):1729-55
references.pmid: 18408885
references.citation: Mol Microbiol. 2004 Feb;51(4):987-1001
references.pmid: 14763975
references.citation: J Biol Chem. 1998 May 15;273(20):12612-22
references.pmid: 9575223
references.citation: Science. 1996 Oct 25;274(5287):546, 563-7
references.pmid: 8849441
references.citation: RNA. 2006 May;12(5):851-61
references.pmid: 16540693
references.citation: Mol Microbiol. 1993 Aug;9(3):521-32
references.pmid: 8412699
references.citation: Trends Biotechnol. 2006 Feb;24(2):55-6
references.pmid: 16380177
references.citation: J Biol Chem. 1991 Apr 25;266(12):7940-9
references.pmid: 1826908
references.citation: J Biol Chem. 1994 Jan 21;269(3):2206-14
references.pmid: 8294477
references.citation: Antimicrob Agents Chemother. 2009 Apr;53(4):1344-52
references.pmid: 19223631
references.citation: Mol Cell Biol. 1988 Jul;8(7):2964-75
references.pmid: 3043201
references.citation: Curr Opin Struct Biol. 2010 Aug;20(4):464-70
references.pmid: 20739007
references.citation: Nat Rev Mol Cell Biol. 2010 Feb;11(2):113-27
references.pmid: 20094052
references.citation: Nature. 2006 Apr 13;440(7086):940-3
references.pmid: 16612385
references.citation: Nature. 2013 Apr 25;496(7446):528-32
references.pmid: 23575629