gal - act osi de lactose
OH O H H H HO H H O OH H HO H H OH H H O HO H O OH
-D-galact ose
-D-glucose H2 O
HO
HO H O OH
H
OH H
O OH OH H
H HO H H
+
OH H
OH HO H H
-D-galact ose
-D-glucose
Structure and binding of tetrameric lac repressor protein
a
b
hinge + helix-turn-helix
IPTG (isopropylthioglucose)
OH HO HO
OH CH2 O S
truncation at hinge
truncation at hinge
Tetramer, with two of the tetrameric units selected
truncation at point of hinge attachment
Lac repressor dimer bound to operator
Headpiece (hinge + HTH motif)
hinge
anti-inducer
A. Looking down DNA helix
B. Rotated 90o around core axis
o-nitrophenylfructose (ONPF)
Contrast inducer-bound and active lac repressor
Lac repressor + ONPF truncated at oligomerization domain
Lac repressor + IPTG truncated at hinge.
NH2 N N O O O P O O OH N N
cyclic AMP (cAMP)
E. coli CAP +cAMP + DNA
HTH motif
cAMP
TFIID = TAFs +
TATA box : is a conserved A T-rich octamer found about 27 bp before the start point of each eukaryotic RNA polymerase II transcription unit; it is involved in positioning the enzyme for correct initiation. A TATA-less promoter : does not have a TATA box in the sequence upstream of its start point. The initiator (Inr) : is the sequence of a pol II promoter between 3 and + 5 and has the general sequence YYANWYY. (C/T C/T A N A/T C/T C/T) It is the simplest possible pol II promoter.
Human TBP complexed at TATA box of DNA
3 'A T A T A C G T A T5 'G C
Eukaryotic Promoters
Saturation mutagenesis of the upstream region of the -globin promoter identifies three short regions (centered at -30, -75, and -90) that are needed to initiate transcription. These correspond to the TATA, CAAT, and GC boxes.
In general. Promoters contain different combinations of TATA boxes, CAAT boxes, GC boxes, and other transcription factor binding sites.
An enhancer contains several transcription factor binding sites (indicated below the sequence. The histogram plots the effect of all mutations that reduce enhancer function to <75% of wild type.
DNA binding motifs Zn fingers Helix-turn-helix Helix-loop-helix Leucine zippers
The zinc finger motif is: -Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-His-
N-terminal
C-terminal
Zn finger fragment bound to DNA
N-terminal
C-terminal
STEROID RECEPTOR
Left: Zn-finger binding domains of dimer complexed with DNA. Right: Ligand binding domain with activator and co-activator bound.
GLUCOCORTICOID RECEPTOR FRAGMENT BOUND TO DNA
Zn binding motif -Cys-X2-Cys-X13-Cys-X2-Cys-
DNA sequence spacing DNA binding N-terminal
finger
C-terminal
Hormone binding and dimerization regions vary from 5-15%
homeodomain
#2 #1 #3, C-terminal
#3 #1, N-terminal Helix-turn-helix binding motif
bHLH DNA binding motif of transcription factor MyoD
Heterodimer of HIF2 alpha and ARNT C-terminal domains
helix 2
helix 1
HN
O
H3 N
N H
OH NH2
prot onat ed arginine
CREB-DNA complex
Leu selected in zipper domain
Arg selected in DNA-binding domain
Human protooncogenes c-Jun:c-Fos heterodimer
Chains: blue = c-Fos, green = c-Jun Residues: red = Leu, purple = Arg
O
Keto-enol tautomerization of Gua N
HN O H2N N N H
O
N N
HN N H2N N H2N N
O H N H
N N
O
OH N
N ON OH N N N keto H form N N HN N NN N N 2 (normal) H2N N N H2N N H2N N N HN N HN N
2 2
OH N
N N
enol form
H3C O N O H OO H N N N N H H O ON N NN H O H N HN N H N N O HN N H HN N N
O H
pairing properties of enol
N N
One round of replication on mismatched strands fixes the mutation.
GTGC+AT
GTGC+AT
Point mutation, transition pyrimidinepyrimidine purinepurine
Point mutation, transversion
GCCG,TAAT,GCTA
pyrimidinepurine purinepyrimidine
nonsense mutation occurs when a termination codon is generated missense mutation occurs when a different amino acid is coded
Change of reading frame following an insertion of G +1 frame shift wild type ATA GCA ACC GGC CTT ACA AAT TC ATA GCA ACC GCC TTA CAA ATT C
Mechanism for frameshift mutations
G-p 3'-G-p-G-p-G-p-G-p-G-p-G-5' 5'-C-p-C p-C-OH p-C
insertion deletion
3'-G-p-G-p G-p-G-p-G-p-G-5' 5'-C-p-C-p-C-OH
Suppressor tRNAs from E. coli
Suppressor supE supP supD supU supF supZ supB supL supN supC supM glyT Type Amber Amber Amber Amber Amber Amber Ochre Ochre Ochre Ochre Ochre Opal Anticodon change CUG --> CUA CAA --> CUA CGA --> CUA CCA --> CUA GUA --> CUA GUA --> CUA UUG --> UUA UUU --> UUA UUU --> UUA GUA --> UUA GUA --> UUA UCC --> UCA tRNA tRNAGln tRNALeu tRNASer tRNATrp tRNATyr tRNATyr tRNAGln tRNALys tRNALys tRNATyr tRNATyr tRNAGly Gene glnV leuX serU trpT tyrT tyrU glnU lysT lysV tyrT tyrU glyT 11-100% Efficiency[1] 0.8-20% 30-100% 6-54%
trpT Opal CCA --> UCA tRNATrp trpT 0.1-30%[2] [1] The suppression efficiency varies for different sites because the mRNA sequence following the nonsense codon influences how well the suppressor tRNA works. [2] Note that this suppressor tRNA can still recognize the wild-type Trp codon as well as the UGA codon.
O CH3 O O O HN O O PO O O O O CH3
HN
O
thymine-thymine dimer
T T
H
H H H H
F p te
error
Human homolog hMsh2
Human homolog hMlh1
Proteinsthatinteractwitheachothersufficientlystronglytoformisolatablecomplexes,andproteinsthathave similarfunctions,havebeengroupedtogetherandprovidedwithacommonbackgroundcolor.Particularly noteworthyisthe10-proteincomplexcalledTFIIH(backgroundcolor=green),whichisessentialforDNArepair andtranscription.
Global genomic nucleotide excision repair pathway (GG-NER)
Transcription-coupled nucleotide excision repair pathway (TC-NER)
Completion of nucleotide excision repair
hTDG glycosylase bound to abasic site remaining after removal of T in a TG mismatch
Abasic sugar, blue
Opposing G, purple Contacts giving hTDG specificity for GpC context are R275 (black) and A274 (green)
human 8-oxoguanine glycosylase (hOGG1) Complex with DNA containing 8-oxoGua
O HN H2N N NH O N
8-oxoGua
8-oxoguanine
Lysine at 249 displaces 8-oxoGua from C1 (arrow)
O
H2N lysine
H2N glutamine
HUMANMITOCHONDRDIALURACILGLYCOSYLASE(UDG),WITH GLYCOSIDICLINKAGEHYDROLYZED
C1'
human alkyladenine glycosylase (AAG) complex with DNA modified by tricyclic base 1,N6- Ade
N N N
6
N N dR
1,N - dAdo
AAG catalyses deglycosylation by a water bridge to Glu
-Elimination
O
P O
O O
B
O O P O O O H
abasic site
OH
H O H
Bas e
Holliday junction
BACTERIAL REPAIR BY RECOMBINATION
Eukaryotic homologues to RecA are characterized in yeast: DMC1 and RAD51