RNA-based transcriptional regulators
RNA molecules have been shown to play a variety of functional roles in biological processes. RNA sequences can adopt 3-D conformations able to regulate transcription. RNA-based transcriptional regulators when recruited to a DNA template act to promote or suppress the transcription of nearby genes. The regulators are thought to act by mimicing the activation domains of protein transcriptional activators. These RNA-based transcriptional regulators may be engineered to regulate transcription based on the binding of a ligand such as a small molecule. RNA sequences of the regulators may be evolved to produce regulators with a greater degree of transcriptional activation or suppression. The RNA-based system of the present invention is useful in studying biological processes and in altering biological process in a therapeutic context.
1. An isolated transcriptional regulator comprising an RNA molecule, wherein the RNA molecule comprises a sequence having at least 80% homology with the sequence set forth as GGAUGCCUAAGCCUCUU (SEQ ID NO: 22), and wherein when the RNA molecule is recruited upstream of a gene, transcription of the gene is increased.
2. The isolated transcriptional regulator of claim 1 , wherein the transcription of the gene is increased 2-fold.
3. The isolated transcriptional regulator of claim 1 , wherein the transcription of the gene is increased 10-fold.
4. The isolated transcriptional regulator of claim 1 , wherein the transcription of the gene is increased 50-fold.
5. The isolated transcriptional regulator of claim 1 , wherein the transcription of the gene is at least as high as a three-hybrid positive control using a natural yeast Gal4 activation domain.
6. The isolated transcriptional regulator of claim 1 , wherein the transcription of the gene is at least 10-fold higher than a three-hybrid positive control using a natural yeast Gal4 activation domain.
7. The isolated transcriptional regulator of claim 1 , wherein the transcription of the gene is approximately 50-fold higher than a three-hybrid positive control using a natural yeast Gal4 activation domain.
8. The isolated transcriptional regulator of claim 1 , wherein the transcription of the gene is approximately two-fold lower than a three-hybrid positive control using natural VP16 activation domain.
9. The isolated transcriptional regulator of claim 1 further comprising an MS2 hairpin that binds to an MS2 coat protein or LexA-MS2 coat protein fusion protein.
10. The isolated transcriptional regulator of claim 1 further comprising an element capable of associating with a DNA-binding protein, wherein the DNA-binding protein is a LexA-MS2 coat protein fusion protein.
11. The isolated transcriptional regulator of claim 1 , wherein the RNA molecule comprises a sequence selected from the group consisting of:
(SEQ ID NO: 23)
m26-10
CGCGGAAGAAUGAUCCCCCAAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 24)
m26-36
CGCGGAAGAAUUCUCCCCCAAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 25)
m26-31
CGCGGAAGAAUGCUCCCCCGAGUGGAUGCCUAAGCCUCAU;
(SEQ ID NO: 26)
m26-9
CGCGGAGGAAUGCUCCCCCGAGUGGAUGCCUAAUCCUCUU;
(SEQ ID NO: 27)
m26-14
CGCGGAAGCAUACUCCCCCGAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 28)
m26-15
CGCGGACCAAUGGUCCCCCAAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 29)
m26-34
CGCGGAAGAUUGCCCCCCCAAGUGGAUGCCUAAACCUCUA;
(SEQ ID NO: 30)
m26-29
CGCGGAAGAUUGUUCCCCCAAGUGGAUGCCUAAACCUCAU;
(SEQ ID NO: 31)
m26-28
CGCGGAAAAAUACUCCCCCAAGUGGAUGCCUAAACCUAUU;
(SEQ ID NO: 32)
m26-17
CGCGGAAGAAUGAUCCCCCAGGUGGAUGCCUAAGCCUCUA;
(SEQ ID NO: 33)
m26-30
CGCGGAACAAUGCUCCCCCAGCUGGAUGCCUAAGCCUCUA;
(SEQ ID NO: 34)
m26-20
CGGGGAAGAAUGCUGCCCAAAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 35)
m26-4
CGCGGAAGAAUGCUGCCCAACCUGGAUGCCUAAACCUCUU;
(SEQ ID NO: 36)
m26-32
CGCGGAAGAAUGCUGCCCAACCUGGAUGCCUAAACCUCUU;
(SEQ ID NO: 37)
m26-13
CUCGGAACAAUGCCCCCCCAAGUGGAUGCCUAAACCUCAU;
(SEQ ID NO: 38)
m26-35
CUCGGAAGAAUUCUCCCGAUAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 39)
m26-16
CGCGGAAGAAGAGUCCCCCGAGUGGAUGCCUAAACCUCUU;
(SEQ ID NO: 40)
m26-18
CGCGGAACAAUGUUGCCCCAAAUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 41)
m26-25
CGCGGAAGAACACUCCCCCACGUGGAUGCCUAAGCCUCCUU;
(SEQ ID NO: 42)
m26-21
CGCGGAAGACUGCAGCCCCAGGUGGAUGCCUAAACCUCGGU;
(SEQ ID NO: 43)
m26-39
CCCGGAAGACUGCAGCCCCAGGUGGAUGCCUAAACCUCUU;
(SEQ ID NO: 44)
m26-33
CGCGGAAGACUUCAGCCCCAAGUGGAUGCCUAAGCCUCUUA;
(SEQ ID NO: 45)
m26-1
CGCGGGUGUAAGCCCCCCCAGGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 46)
m26-23
CGCGUAAACAUGCGGCCCCAAGUGGAUGCCUAAGCCUCCU;
(SEQ ID NO: 47)
m26-24
CGCGGAAAGAUGAUCACCCGAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 48)
m26-11
CGCGCGAGUAUACUCCCCCAAGCGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 49)
m26-19
CGCAGAAGAUACCUCCCCCAAGUGGAUGCCUAAGCCUCAU;
(SEQ ID NO: 50)
m26-26
AACAGAAGAAUACUCCCCCAAGUGGAUACCUAAGCAUCUU;
(SEQ ID NO: 51)
m26-37
CGCGGAAAAAUUCUUCCCCCAAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 52)
m26-38
CGCGGAAAAAUUCUUCCCCCAAGUGGAUGCGUAAGCCUCUU;
(SEQ ID NO: 53)
m26-7
CGCGGAAGAACGCUCCCCGACGUGGAUGCCUAUUGUCCUU;
and
(SEQ ID NO: 54)
con-
CGCGGAAGAAUGCUCCCCCAAGUGGAUGCCUAAGCCUCUU.
sensus
12. The isolated transcriptional regulator of claim 1 , wherein the RNA molecule comprises the sequence:
consensus
(SEQ ID NO: 54)
CGCGGAAGAAUGCUCCCCCAAGUGGAUGCCUAAGCCUCUU.
13. The isolated transcriptional regulator of claim 1 , wherein the RNA molecule comprises the sequence:
(SEQ ID NO: 30)
m26-29
CGCGGAAGAUUGUUCCCCCAAGUGGAUGCCUAAACCUCAU.
14. The isolated transcriptional regulator of claim 1 , wherein the RNA molecule comprises the sequence:
(SEQ ID NO: 48)
m26-11
CGCGCGAGUAUACUCCCCCAAGCGGAUGCCUAAGCCUCUU.
15. The isolated transcriptional regulator of claim 1 , wherein the RNA molecule comprises a sequence responsible for transcriptional regulation that ranges from 20-50 bases in length.
16. The isolated transcriptional regulator of claim 1 , wherein the gene is HIS 3.
17. The isolated transcriptional regulator of claim 1 , wherein the gene is LacZ.
18. The isolated transcriptional regulator of 1 comprising the tetramethylrosamine (TMR)-binding aptamer of sequence:
CGACUGGCGAGAGCCAGGUAACGAAU.
(SEQ ID NO: 70)
19. The isolated transcriptional regulator of claim 1 further comprising an RNA molecule comprising a sequence selected from the group consisting of:
(SEQ ID NO: 61)
CGACUGGCGAGAGCCAGGUAACGAAUCCUGGGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 62)
CGACUGGCGAGAGCCAGGUAACGAAUCCGGGCAGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 63)
CGACUGGCGAGAGCCAGGUAACGAAUCCUUACGCGGAUGCCUAAGCCUCU
U;
(SEQ ID NO: 64)
CGACUGGCGAGAGCCAGGUAACGAAUCCUAACCGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 65)
CGACUGGCGAGAGCCAGGUAACGAAUCCCAACCGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 66)
CGACUGGCGAGAGCCAGGUAAAGAAUCCUAACCGGAUGCCUAAGCCUCUU;
and
(SEQ ID NO: 67)
CGACUGGCGAGAGCCAGGUACCGAAUCCUAACCGGAUGCCUAAGCCUCUU.
20. An isolated vector encoding the transcriptional regulator of claim 1 .
21. The isolated vector of claim 20 further comprising an RNase P RNA gene (RPR) promoter.
22. The isolated vector of claim 20 further comprising an RNase P RNA gene (RPR) terminator.
23. The isolated vector of claim 20 , wherein the vector is a plasmid.
24. The isolated vector of claim 23 , wherein the plasmid can be propagated in yeast.
25. The isolated vector of claim 23 , wherein the plasmid is derived from yeast shuttle vector pIIIa/MS2.
26. The isolated vector of claim 23 , wherein the plasmid includes marker genes.
27. The isolated transcriptional regulator of claim 1 , wherein the RNA molecule comprises a sequence having at least 80% homology with a sequence selected from the group consisting of:
CCCAAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 60)
CCCAAGCGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 69)
and
CCCAACCGGAUGCCUAAGCCUCUU.
(SEQ ID NO: 71)
28. The isolated transcriptional regulator of claim 1 , wherein the RNA molecule comprises a sequence having at least 90% homology with a sequence selected from the group consisting of:
CCCAAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 60)
CCCAAGCGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 69)
and
CCCAACCGGAUGCCUAAGCCUCUU.
(SEQ ID NO: 71)
29. The isolated transcriptional regulator of claim 1 , wherein the RNA molecule comprises a sequence having at least 95% homology with a sequence selected from the group consisting of:
CCCAAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 60)
CCCAAGCGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 69)
and
CCCAACCGGAUGCCUAAGCCUCUU.
(SEQ ID NO: 71)
30. The isolated transcriptional regulator of claim 1 , wherein the RNA molecule comprises a sequence having up to 1 nucleotide mismatch with a sequence selected from the group consisting of:
CCCAAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 60)
CCCAAGCGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 69)
and
CCCAACCGGAUGCCUAAGCCUCUU.
(SEQ ID NO: 71)
31. The isolated transcriptional regulator of claim 1 , wherein the RNA molecule comprises a sequence selected from the group consisting of:
CCCAAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 60)
CCCAAGCGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 69)
and
CCCAACCGGAUGCCUAAGCCUCUU.
(SEQ ID NO: 71)
32. An isolated RNA molecule that functions as a protein transcriptional activation domain, wherein the RNA molecule comprises a sequence having at least 80% homology with the sequence set forth as GGAUGCCUAAGCCUCUU (SEQ ID NO: 22).
33. An isolated transcriptional regulator comprising an RNA molecule comprising a sequence selected from the group consisting of:
(SEQ ID NO: 23)
m26-10
CGCGGAAGAAUGAUCCCCCAAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 24)
m26-36
CGCGGAAGAAUUCUCCCCCAAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 25)
m26-31
CGCGGAAGAAUGCUCCCCCGAGUGGAUGCCUAAGCCUCAU;
(SEQ ID NO: 26)
m26-9
CGCGGAGGAAUGCUCCCCCGAGUGGAUGCCUAAUCCUCUU;
(SEQ ID NO: 27)
m26-14
CGCGGAAGCAUACUCCCCCGAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 28)
m26-15
CGCGGACCAAUGGUCCCCCAAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 29)
m26-34
CGCGGAAGAUUGCCCCCCCAAGUGGAUGCCUAAACCUCUA;
(SEQ ID NO: 30)
m26-29
CGCGGAAGAUUGUUCCCCCAAGUGGAUGCCUAAACCUCAU;
(SEQ ID NO: 31)
m26-28
CGCGGAAAAAUACUCCCCCAAGUGGAUGCCUAAACCUAUU;
(SEQ ID NO: 32)
m26-17
CGCGGAAGAAUGAUCCCCCAGGUGGAUGCCUAAGCCUCUA;
(SEQ ID NO: 33)
m26-30
CGCGGAACAAUGCUCCCCCAGCUGGAUGCCUAAGCCUCUA;
(SEQ ID NO: 34)
m26-20
CGGGGAAGAAUGCUGCCCAAAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 35)
m26-4
CGCGGAAGAAUGCUGCCCAACCUGGAUGCCUAAACCUCUU;
(SEQ ID NO: 36)
m26-32
CGCGGAAGAAUGCUGCCCAACCUGGAUGCCUAAACCUCUU;
(SEQ ID NO: 37)
m26-13
CUCGGAACAAUGCCCCCCCAAGUGGAUGCCUAAACCUCAU;
(SEQ ID NO: 38)
m26-35
CUCGGAAGAAUUCUCCCGAUAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 39)
m26-16
CGCGGAAGAAGAGUCCCCCGAGUGGAUGCCUAAACCUCUU;
(SEQ ID NO: 40)
m26-18
CGCGGAACAAUGUUGCCCCAAAUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 41)
m26-25
CGCGGAAGAACACUCCCCCACGUGGAUGCCUAAGCCUCCUU;
(SEQ ID NO: 42)
m26-21
CGCGGAAGACUGCAGCCCCAGGUGGAUGCCUAAACCUCGGU;
(SEQ ID NO: 43)
m26-39
CCCGGAAGACUGCAGCCCCAGGUGGAUGCCUAAACCUCUU;
(SEQ ID NO: 44)
m26-33
CGCGGAAGACUUCAGCCCCAAGUGGAUGCCUAAGCCUCUUA;
(SEQ ID NO: 45)
m26-1
CGCGGGUGUAAGCCCCCCCAGGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 46)
m26-23
CGCGUAAACAUGCGGCCCCAAGUGGAUGCCUAAGCCUCCU;
(SEQ ID NO: 47)
m26-24
CGCGGAAAGAUGAUCACCCGAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 48)
m26-11
CGCGCGAGUAUACUCCCCCAAGCGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 49)
m26-19
CGCAGAAGAUACCUCCCCCAAGUGGAUGCCUAAGCCUCAU;
(SEQ ID NO: 50)
m26-26
AACAGAAGAAUACUCCCCCAAGUGGAUACCUAAGCAUCUU;
(SEQ ID NO: 51)
m26-37
CGCGGAAAAAUUCUUCCCCCAAGUGGAUGCCUAAGCCUCUU;
(SEQ ID NO: 52)
m26-38
CGCGGAAAAAUUCUUCCCCCAAGUGGAUGCGUAAGCCUCUU;
(SEQ ID NO: 53)
m26-7
CGCGGAAGAACGCUCCCCGACGUGGAUGCCUAUUGUCCUU;
and
(SEQ ID NO: 54)
con-
CGCGGAAGAAUGCUCCCCCAAGUGGAUGCCUAAGCCUCUU.
sensus
34. The isolated transcriptional regulator of claim 33 comprising an RNA molecule comprising the sequence of:
consensus
(SEQ ID NO: 54)
CGCGGAAGAAUGCUCCCCCAAGUGGAUGCCUAAGCCUCUU.
35. The isolated transcriptional regulator of claim 33 comprising an RNA molecule comprising the sequence of:
(SEQ ID NO: 30)
CGCGGAAGAUUGUUCCCCCAAGUGGAUGCCUAAACCUCAU (m26-12).
36. The isolated transcriptional regulator of claim 33 further comprising at least one MS2 hairpin.
37. The isolated transcriptional regulator of claim 33 further comprising at least two MS2 hairpins.
38. The isolated transcriptional regulator of claim 33 further comprising an Iron Response Element (IRE).
39. The isolated transcriptional regulator of claim 33 further comprising a fusion of an Iron Response Element (IRE) and an MS2 hairpin.
40. The isolated transcriptional regulator of claim 33 further comprising a RPR terminator.
41. The isolated transcriptional regulator of claim 33 , further comprising a DNA binding moiety, wherein the DNA binding moiety is a LexA-MS2 fusion protein.
42. A transcriptional regulatory system comprising:
an isolated transcriptional regulator comprising an RNA molecule comprising a sequence having at least 80% homology with the sequence set forth as GGAUGCCUAAGCCUCUU (SEQ ID NO: 22);
a DNA-binding protein; and
a DNA template.
43. The transcriptional regulatory system of claim 42 further comprising transcriptional machinery.
44. The transcriptional regulatory system of claim 43 , wherein the transcriptional machinery comprises RNA polymerase.