Substituting this into Eq. (A.27) yields
[D]
2 + [D]2
KDT = [R], (A.42)
which we can solve for [D]:
[D] =1 4
−KDT + q
KDT2 + 16KDT[R])
(A.43)
where the positive root is chosen because [D] must be positive and the discriminant is always positive.
However we want [T], not [D]; so we again use Eq. (A.27) to say that
[T] = [R]−[D]
2 = [R]−1 8
−KDT+ q
KDT2 + 16KDT[R])
. (A.44)
By substituting this into Eq. (A.39) we obtain our final partition function of
Z= 1 + [R]
Kid
+[R]
K1
+ [R]2 K1Kid
+[R]Jloop 2K1Kid
1− 1
8[R]
−KDT + q
KDT2 + 16KDT[R])
. (A.45)
Since the looping probability is the weight of the looped state divided by the partition function, we have our final result that
ploop, dimers=
[R]Jloop
2K1Kid
1 + K8[R]DT −8[R]1 p
KDT2 + 16KDT[R]) 1 + K[R]
id+[R]K
1 +K[R]2
1Kid +[R]J2K loop
1Kid
1 +K8[R]DT −8[R]1 p
KDT2 + 16KDT[R]) .
(A.46)
to form tetramers. This will lead to a fraction of dimers,ν, that is constant with the total repressor concentration [R]. (As noted in Chapter 2, we could consider a third case, in which a fraction of monomers that are damaged such that when incorporated into a tetramer they result in a head that is unable to bind DNA. We will not consider this case here but it is well within the scope of scenarios that can be captured by the class of models presented here.)
Note that since we assumed in the previous section that the binding of dimers and tetramers to the DNA did not affect the equilibrium reaction between tetramers and dimers in solution, we can start with Eq. (A.39), since the derivations for the two cases are the same up to this point.
We will define the concentration of repressors in tetrameric form as
[T] = (1−ν)[R], (A.47)
where ν is the fraction that are dimers. Again we are considering here the case where the dimeric fraction is constant with the total concentration. Then because [D]2 + [T] = [R], we must defineν as
ν = [D]
2[R], (A.48)
so that [D]/2+[T] = 2ν[R]/2+(1−ν)[R] = [R]. We can now use this expression for [T] in Eq. (A.39), so that when we form the looping probability we obtain
ploop, dimers =
(1−ν)[R]J 2K1Kid
1 + K[R]
id+[R]K
1 +K[R]2
1Kid +(1−ν)[R]J2K
1Kid
. (A.49)
Note that this is the same result we obtained in the previous section, if we use the expression for [D] in Eq. (A.43) in the definition ofν as 2[R][D], except that hereν is a scalar, not a function of [R].
Appendix B
DNAs
B.1 Constructs containing E8 and 601TA
The E8- and TA-containing constructs discussed in Chapters 3 and 4 are PCR products of plasmids pZS25’ Oid-E/T(89-116)-O1−45-YFP, where “E/T(89-116)” indicates that the sequence of the loop is either from the random E8 sequence or the 601TA sequence from [85] and has a length of 89 to 116 bp. The original constructs used in [115] and [120] (lengths 89, 94, and 100 bp) were constructed by site-directed mutagenesis as described in [115]. Jonathan Widom kindly provided the E8 and TA sequences used in [85], which are a subset of those used here and from which the other E8 and TA lengths were derived. QuikChange site-directed mutagenesis (Agilent Technologies) was used to make the operator changesOid toO1 andOid to O2, additional loop lengths, and the promoter-containing constructs. Linear labeled DNAs used in tethering assays were created by PCR with primers labeled at the 5’ ends with digoxigenin (forward primers) or biotin (reverse primers) (Eurofins MWG Operon); a PCR of the pZS25’ plasmids resulted in approximately 450 bp tethers
Name Sequence
O1 AATTGTGAGCGGATAACAATT
O2 GGTTGTTACTCGCTCACATTT
O3 GGCAGTGAGCGCAACGCAATT
Oid AATTGTGAGCGCTCACAATT
Table B.1: Sequences of the three naturally occurring Lac repressor operatorsO1,O2, andO3, and of the synthetic Oid(“Oideal”) operator. All sequences are 5´ to 3´ and are from [94]. Note thatOidis perfectly symmetric about its midpoint, whereas the naturally occurring, weaker operators are only pseudo-symmetric, withO1being the strongest, O2 weaker, andO3 the weakest. In this work, the loop is to the 3´ end of the appropriate (Oid/O1/O2) operator sequence shown here; theO1 that is constant in all constructs (nearest to the bead) has the loop 5´ to the sequence given here.
E89: GGCCG––– ––– –––GCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGAC ––– ––– ––– ––– ––– ––– C E90: GGCCG––– ––– ––TGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGAC ––– ––– ––– ––– ––– ––– C E91: GGCCG––– ––– –CTGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGAC ––– ––– ––– ––– ––– ––– C E92: GGCCG––– ––– –CTGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACG–– ––– ––– ––– ––– ––– C E93: GGCCG––– ––– –CTGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGT– ––– ––– ––– ––– ––– C E94: GGCCG––– ––– ––– ––TGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGTCCGCC –– ––– ––– ––– C E95: GGCCGG –––––GCTGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGT– ––– ––– ––– ––– ––– C E96: GGCCG ––––AGGCTGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGT– ––– ––– ––– ––– ––– C E97: GGCCG ––––AGGCTGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGTC––– ––– ––– ––– ––– C E98: GGCCG ––––AGGCTGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGTCC–– ––– ––– ––– ––– C E99: GGCCG –––GAGGCTGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGTCC–– ––– ––– ––– ––– C E100: GGCCG –––GAGGCTGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGTCCG– ––– ––– ––– ––– C E101: GGCCG ––– ––– ––– GCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGTCCGCCAGCCG––– ––– C E102: GGCCG ––– ––– ––– GCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGTCCGCCAGCCGA–– ––– C E103: GGCCG ––– ––– ––– GCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGTCCGCCAGCCGAC– ––– C E104: GGCCG ––– ––– ––– GCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGTCCGCCAGCCGACG––– C E105: GGCCG ––– ––– ––TGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGTCCGCCAGCCGACG––– C E106: GGCCG ––– ––– –CTGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGTCCGCCAGCCGACG––– C E107: GGCCG ––– ––– GCTGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGTCCGCCAGCCGACG––– C E108: GGCCG ––– –– GGCTGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGTCCGCCAGCCGACG––– C E109: GGCCG ––– –AGGCTGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGTCCGCCAGCCGACG––– C E116: GGCCGGCGGAGGCTGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACGTCCGCCAGCCGACGACGC
T89: ––– ––– ––– –GGCCG––– ––– –GGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGT–– ––– ––– ––– ––– C T90: ––– ––– ––– –GGCCG––– –––TGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGT–– ––– ––– ––– ––– C T91: ––– ––– ––– –GGCCG––– ––TTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGT–– ––– ––– ––– ––– C T92: ––– ––– ––– –GGCCG––– ––TTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC– ––– ––– ––– ––– C T93: ––– ––– ––– –GGCCG––– ––– –GGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCT– ––– ––– ––– C T94: ––– ––– ––– –GGCCG––– ––– –GGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCTA––– ––– ––– C T95: ––– ––– ––– –GGCCG––– –ATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTC–– ––– ––– ––– C T96: ––– ––– ––– –GGCCG–––AATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTC–– ––– ––– ––– C T97: ––– ––– ––– –GGCCG––TAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTC–– ––– ––– ––– C T98: ––– ––– ––– –GGCCG––TAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCT– ––– ––– ––– C T99: ––– ––– ––– –GGCCG––TAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCTA––– ––– ––– C T100: ––– ––– ––– –GGCCG––TAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCTAG–– ––– ––– C T101: ––– ––– ––– –GGCCG––TAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCTAGG– ––– –––C T102: ––– ––– ––– –GGCCG––TAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCTAGGC ––– –––C T103: ––– ––– ––– –GGCCG––TAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCTAGGCA–– ––– C T104: ––– ––– ––– –GGCCG–TTAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCTAGGCA–– ––– C T105: ––– ––– ––– –GGCCG–TTAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCTAGGCAC– ––– C T106: ––– ––– ––– –GGCCG–TTAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCTAGGCACG ––– C T107: ––– ––– ––– –GGCCG–TTAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCTAGGCACGT–– C T108: ––– ––– ––– –GGCCG–TTAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCTAGGCACGTG– C T109: ––– ––– ––– –GGCCG–TTAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCTAGGCACGTGTC T116: CCGGTGCTAAGGCCGCTTAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCTAGGCAC––––C
601TA: ctggagatacCCGGTGCTAAGGCCGCTTAATTGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTCTCTAGG CACGTGT–aagatatatacatcctgtgcatgta
Figure B.1: Sequences of the no-promoter E8 and TA constructs used in this work (Fig. 4.2(A–C)). All sequences are 5´ to 3´ and are listed such that theOidoperator (or O1 or O2 operator in the case of the E894 sequence) is immediately 5´ of these sequences, andO1 is immediately 3´. Bolded sequence labels indicate constructs examined by cyclization in [85], which were incorporated into the pZS25’ plasmid by Hernan Garcia; the rest were designed and created by Stephanie Johnson. In the top section containing the E8 sequences, dashes indicate bases missing relative to the 116 bp E8 sequence listed at the bottom of that section. In the bottom section containing the TA sequences, dashes indicate bases missing relative to the full 154 bp 601TA sequence (provided to us by Jon Widom; see also [86]) listed below the TA sequences; in that 601TA sequence, the dash indicates where a C has been inserted at the end of all of the TA sequences used in both cyclization [58, 85] and in the looping work presented here. Upper-case letters in the full 601TA sequence indicate the region from which all TA sequences in this work were derived. The 601TA sequence is so named because of the TA dinucleotide steps which occur every 10 bp and which are thought to confer its affinity for nucleosome formation [14]; these TA steps have been highlighted in red. Note that the E8 sequence also has several TA steps spaced 10 bp apart; however this pattern does not repeat across the entire sequence as it does in the 601TA sequence, nor does the E8 sequence have other characteristics of the 601TA sequence such as GC pairs between the TA pairs which are also supposed to be important for its particular properties [14, 52].
E92: –– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– –CTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E93: –– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– CCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E94: –– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––GCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E95: –– ––– ––– ––– ––– ––– ––– ––– ––– ––– –CGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E96: –– ––– ––– ––– ––– ––– ––– ––– ––– ––– TCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E97: –– ––– ––– ––– ––– ––– ––– ––– ––– ––ATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E101: –– ––– ––– ––– ––– ––– ––– ––– –ATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E103: –– ––– ––– ––– ––– ––– ––– ––TTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E105: –– ––– ––– ––– ––– ––– –––TTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E114: –– ––– ––– ––– TAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E115: –– ––– ––– ––GTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E116: –– ––– ––– –CGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E117: –– ––– ––– GCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E118: –– ––– ––TGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E119: –– ––– –CTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E120: –– ––– GCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E121: –– ––GGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E122: –– –CGGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E123: –– CCGGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC E124: G–CCGGCTGCGTAGAACTACTTTTATTTATCGCCTCCACGGTGCTGATCCCCTGTGCTGTTGGCCGTGTTATCTCGAGTTAGTACGACC
T92: –– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– –ACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T93: –– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––– AACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T94: –– ––– ––– ––– ––– ––– ––– ––– ––– ––– ––AAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T95: –– ––– ––– ––– ––– ––– ––– ––– ––– ––– –TAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T96: –– ––– ––– ––– ––– ––– ––– ––– ––– ––– TTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T97: –– ––– ––– ––– ––– ––– ––– ––– ––– ––CTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T101: –– ––– ––– ––– ––– ––– ––– ––– –ACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T103: –– ––– ––– ––– ––– ––– ––– ––GCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T105: –– ––– ––– ––– ––– ––– –––TAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T106: –– ––– ––– ––– ––– ––– ––CTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T107: –– ––– ––– ––– ––– ––– –TCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T108: –– ––– ––– ––– ––– ––– CTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T114: –– ––– ––– ––– AGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T115: –– ––– ––– ––TAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T116: –– ––– ––– –GTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T117: –– ––– ––– CGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T118: –– ––– –– TCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T119: –– ––– –GTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T120: –– –––GGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T121: –– ––GGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T122: –– –CGGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T123: –– CCGGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC T124: G–CCGGGTCGTAGCAAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCTACCGCGTTTTAACCGCCAATAGGATTACTTACTAGTC
Figure B.2: Sequences of the with-promoter E8 and TA constructs used in this work (Fig. 4.2(D–F)). All sequences are 5´ to 3´ and are listed such that theOidoperator is immediately 5´ of these sequences, andO2is immediately 3´
(butO2 is the reverse complement of the sequence given in Table B.1). ThelacUV5 promoter is to the 3´ end, before theO2operator; its sequence is TTTACAATTAATGCTTCCGGCTCGTATAATGTGTGG. As in Fig. B.1, TA steps have been highlighted in red. Dashes indicated bases missing from the 89 bp no-promoter equivalents shown in the previous figure.
PolyA105:*************ACCTTGTATTGTATTTCCTTTGCGTGATGAAAAAAAAACTGAAAAAGAGAAAAATAAGAAAATCTTCTAGAACGTTCCGAAACAGGAC–gtgctgatcccctgtgc––*
PolyA106:*************ACCTTGTATTGTATTTCCTTTGCGTGATGAAAAAAAAACTGAAAAAGAGAAAAATAAGAAAATCTTCTAGAACGTTCCGAAACAGGAC–gtgctgatcccctgtgct–*
PolyA107:*************ACCTTGTATTGTATTTCCTTTGCGTGATGAAAAAAAAACTGAAAAAGAGAAAAATAAGAAAATCTTCTAGAACGTTCCGAAACAGGACggtgctgatcccctgtgct–*
PolyA108:*************ACCTTGTATTGTATTTCCTTTGCGTGATGAAAAAAAAACTGAAAAAGAGAAAAATAAGAAAATCTTCTAGAACGTTCCGAAACAGGACggtgctgatcccctgtgctg*
*
PolyA105(prom):*–––*–––*–––*–––*–––*–––*–TTGCGTGATGAAAAAAAAACTGAAAAAGAGAAAAATAAGAAAATCTTCTAGAACGTTCCGAAACAGGAC*
PolyA106(prom):*–––*–––*–––*–––*–––*–––*TTTGCGTGATGAAAAAAAAACTGAAAAAGAGAAAAATAAGAAAATCTTCTAGAACGTTCCGAAACAGGAC*
PolyA107(prom):*–––*–––*–––*–––*–––*––CTTTGCGTGATGAAAAAAAAACTGAAAAAGAGAAAAATAAGAAAATCTTCTAGAACGTTCCGAAACAGGAC*
PolyA108(prom):*–––*–––*–––*–––*–––*–CCTTTGCGTGATGAAAAAAAAACTGAAAAAGAGAAAAATAAGAAAATCTTCTAGAACGTTCCGAAACAGGAC*
*
Figure B.3: Sequences of the poly(dA:dT)-rich sequences used in Chapter 4, which are derived from the nucleosome-* free region of theS. cerevisiaepromoter given in Fig. 4E of [149]. This 88 bp poly(dA:dT)-rich sequence is shown in capital letters, with stretches of more than four consecutive A’s highlighted in green, where we have chosen to define an A-tract as 4 or more A’s in a row because this is the shortest length that shows special structural properties under a variety of methods [148]. The top section lists no-promoter sequences; the bottom section, sequences to which the 36 bplacUV5 promoter was added to the loop as in Fig. B.2. As in that figure and Fig. B.1, theOidoperator is to the left of all of these sequences, and theO1operator (for the no-promoter sequences) or the promoter and thenO2
(for the with-promoter sequences) to the right. The DNA flanking the loop region are the same as those for the E8 and TA constructs (lengths given in Fig. 1.4). As the poly(dA:dT)-rich region of [149] is only 88 bp, the no-promoter constructs were padded with a portion of the E8 sequence (which should be a random sequence); these bases are shown in lower-case letters. Dashes in the no-promoter construct indicate where bases were removed relative to the 108 bp construct. In the with-promoter construct, dashes indicate bases removed relative to the 88 bp sequence from [149].
(see Fig. 1.4 for flanking DNA lengths). Primer sequences can be found in Table 3 of [115]. The PCR product was gel purified using a QIAquick Gel Extraction Kit (Qiagen), and the concentration determined by quantitative gel electrophoresis.
Table B.1 gives the sequences of the three naturally occurring operators of the lac operon and the strong synthetic operator Oid used in some of the work discussed here. Figures B.1 and B.2 shows the E8 and TA sequences that form the loops in the constructs discussed in Chapters 3 and 4.
All constructs were verified by sequencing (Laragen).