P1248802078LEvdz - PowerPoint PPT Presentation

Title: P1248802078LEvdz


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Problems Session?
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• The lac repressor bound to operator sequences and
  the CAP-cAMP in complex with its 30 bp binding
  site. The TATA box and -35 region of the
  promoter are also indicated.

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Catabolite repression happens when glucose (a
catabolite) levels are high.

• Then cyclic AMP is inhibited from forming.
• When glucose levels drop, more cAMP forms.
• cAMP binds to a protein called CAP (catabolite
  activator protein), which is then activated to
  bind to the CAP binding site.
• This activates transcription, perhaps by
  increasing the affinity of the site for RNA
  polymerase.
• This phenomenon is called catabolite repression,

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Suggested readings on regulation/dna bp Voet
pp 1237-1253 Problems 2, 4 Heres a quiz on the
lac operon http//www.bio.davidson.edu/courses/
movies.html
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Figure 31-39 A genetic map of the E. coli trp
operon indicating the enzymes it specifies and
the reactions they catalyze.
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Figure 31-40 The base sequence of the trp
operator. The nearly palindromic sequence is
boxed and its 10 region is overscored.
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Figure 31-41 The alternative secondary structures
of trpL mRNA.
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Figure 31-42a Attenuation in the trp operon. (a)
When tryptophanyltRNATrp is abundant, the
ribosome translates trpL mRNA.
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Figure 31-42b Attenuation in the trp operon. (b)
When tryptophanyltRNATrp is scarce, the ribosome
stalls on the tandem Trp codons of segment 1.
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Table 31-3 Amino Acid Sequences of Some Leader
Peptides in Operons Subject to Attentuation.
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Figure 31-43 The structure of the 5 cap of
eukaryotic mRNAs.
Page 1255
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Figure 31-46 An electron micrograph and its
interpretive drawing of a hybrid between the
antisense strand of the chicken ovalbumin gene
and its corresponding mRNA.
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Figure 31-47 The sequence of steps in the
production of mature eukaryotic mRNA as shown for
the chicken ovalbumin gene.
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Figure 31-48 The consensus sequence at the
exonintron junctions of vertebrate pre-mRNAs.
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Figure 31-49 The sequence of transesterification
reactions that splice together the exons of
eukaryotic pre-mRNAs.
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Table 31-4 Types of Introns.
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DNA Binding Proteins
How does a repressor find its operator in a sea
of other sequences?
It is not enough just for the regulatory protein
to recognize the correct DNA site. The protein
must also find it rapidly and bind to it
sufficiently tightly to discriminate it from the
millions of competing and overlapping
nonspecific sites that are explored in the
course of specific target localization.
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Workshop http//www.rpi.edu/bellos/DNA-PROTEIN20
INTERACTIONS.ppt
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Association constants lac repressor DNA to
R-DNA complex Repressor lac operator 1-2 X
1013 M-1 other DNA 2-3 X 106 M-1 (specificity
KA(s)/KA(non-specific) 107) Repressor bound to
inducer lac operator 2 X 1010 M-1--or some
references suggest this is even lower other DNA
2 X 106 M-1 When repressor is bound to
allosteric regulator (allolactose in this case)
non-specific binding competes more effectively
with specific binding.
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How a repressor recognizes and binds to an
operator The interaction between repressor and
operator is often taken as a paradigm for
sequence-specific DNA-protein interactions.
Each regulatory protein in E. coli must select
its operator site (or sites) from among the five
million or so base pairs of DNA in the cell.
Examples?
For this organism, an operator (or any other cis
acting site) must be at least 11-12 bases long
in order to form a site that reoccurs at random
less than once per genome.
Accordingly, regulatory proteins in E. coli bind
tightly to specific DNA sequences that are about
15-20 base pairs long.
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Operator Sequence and Structure
A large number of operator sites have been
identified and their DNA sequence has been
determined.
One feature that is common to all operators is an
imperfect two-fold axis of symmetry.

• A perfectly symmetrical sequence is shown below.
• gt---- G C C A T G C G C A T G G C ----gt
• lt---- C G G T A C G C G T A C C G ----lt

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Cap binding site
Link to view structure
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Lac repressor
lac operator binding site for the lac repressor
protein (lac I gene product)
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Structure of Regulatory Proteins Many
DNA-binding regulatory proteins share features in
common that reflect a common mode of DNA
binding. Some of these features are
(1) The active binding unit is a dimer of two
identical globular polypeptide chains oriented
oppositely in space to give a molecule with a
two-fold axis of symmetry
phage lambda cI repressor protein alpha helical
region in contact with the major groove is in
red.
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(2) The critical contacts between the protein
and the DNA are made by adjacent a helices
located at the binding face of each monomer. The
helices are connected by a turn in the protein
secondary structure. This helix-turn-helix
motif is common to many regulatory proteins.
HTH
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Cro
Why do the recognition helices contact the major
groove? What determines the specificity of
interaction?
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Binding motifs http//www.umass.edu/molvis/freich
sman/Site642/page_dnab/menu.html again http//ww
w.web-books.com/MoBio/Free/Ch4F2.htm4F1
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AH bond acceptor DH bond donor
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One of the most common DNA-protein interactions.
Because of its specific geometry of H-bond
acceptors, guanine can be unambiguously
recognized by the side chain of arginine
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Stereo view for phage lambda repressor
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• Specificity of protein-DNA interaction of due to
• ability of amino acid side chains in the
  recognition helix to form hydrogen bonds with
  specific bases in its cis-acting site
• multiple complementary interactions between the
  protein and the DNA that are dependent on the
  deformation of the helix and which increase the
  number of contact points

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Main features of interactions between DNA and
the helix-turn-helix motif of DNA binding
proteins
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Phage Lambda
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Binding tutuorial
http//www.biochem.arizona.edu/classes/bioc462/462
a/NOTES/ Nucleic_Acids/prodna.html
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Important Points a handshake leads to a bear-hug
Specific recognition of DNA targets by the
helix-turn-helix motif involves interactions
between sides of the recognition helix and bases
in the major groove of the DNA
But, specific recognition of DNA sequences is to
a large extent governed by other interactions
within complementary surfaces between the protein
and the \
These interactions frequently involve H-bonds
from protein main-chain atoms to the DNA backbone
in both the major and the minor groove and are
dependent on the sequence-specific deformability
of the target DNA
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DNA deformation induced by protein-binding.
The ease with which a stretch of DNA can be
deformed can affect the affinity of protein
binding to a specific sequence
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CAP binding to its cis-acting site cAMP binding
domain in blue red -- DNA phosphates whose
ethylation interferes with cap binding
blue hypersenstivie to DNase I-- these
phosphates bridge the cap-induced where the
minor groove has been widened
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Here the lac repressor tetramer is shown binding
to two operators. Each dimer contacts one
operator (either dark or light blue). The
operators are 21 bases long.
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MVA Fig.21.15
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No direct H-bonding with bases!
All specific H-bonds occur via bridging water
molecules!
Only direct contacts are H-bonds to the
phosphate backbone!!!!
Yet mutations of these non-contact bases alter
binding specificity
This suggests that the operator assumes a
sequence-specific conformation that makes
favorable contacts with the repressor known as
Indirect Readout
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When tryptophan is added to crystals of
aporepressor, the crystals shatter. When the
tryptophan wedges itself into the protein, it
changes the shape of the protein enough to break
the lattices of the crystal The orientation of
the recognition helix shifts when tryptophan is
bound.
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trp repressor (HTH allosteric)
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trp repressor (HTH allosteric)
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His
Cys
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?
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mutations that affect DNA binding are oncogenic
minor groove
major groove
p53 DNA binding domain
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bZip
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bZip homo- and heterodimers