molecular ex 2 Flashcards
1
Q
Labeled tracers are…
A
radioactive.
ph32 used to detect DNA,
sulfur35 to detect proteins.
2
Q
autoradiography
A
detects radioactive compounds using photographic emulsion (x ray), specifically tracers and labels
3
Q
nonradioactive tracers
A
use enzymes instead of radioactivity, uses multiplier effect of an enzyme to generate a signal
4
Q
Nonradioactive tracers may use _____ for dUTP along with avidin because it has a natural affinity for avidin
A
Biotin
5
Q
nonradioactive tracers use a phosphate substrate
A
to inhibit light from fluorescing, followed by a phosphotase (attached to desired affinity) that will remove those phosphate groups to enable light
6
Q
Nucleic acid hybridization
A
one single stranded nucleic acid forms a double helix with another single strand of complementary base sequence
7
Q
Southern hybridization
A
detects DNA signals using probe thats complementary to target (for ex, biotin), the target DNA must be separated, then the probe can be used.
8
Q
Southern blots (occurs after gel electro)
A
-pos charge, can bind to neg charged
-can only bind to double strands
-DNA is separated on gel
-gel is treated w alkali to denature DNA, resulting in ss DNA that binds to filter
-pieces are bound to nitrocellulose filter by electrophor.
-comp cDNA probe is applied to filter (LAST STEP), positive band now detectable where hybrid between probe and DNA happened.
9
Q
southern blot bands are seen with
A
x ray film or autoradiography
10
Q
southern blot bands could show
A
multiple genes or several restriction sites in gene
11
Q
__________ can be used in forensic labs to detect people using DNA
A
southern blots
12
Q
mini satellite DNA
A
sequence of bases unique to every person repeated several times, called DNA fingerprint (not used anymore)
13
Q
Parts of DNA fingerprint pattern can be inherited (Mendelian) (t/f)
A
True
14
Q
DNA fingerprinting uses
A
set of probes to give set of simple patterns, and uses probes for just a single locus
15
Q
Immunoblots AKA Western blots are similar to
A
southern blots
16
Q
Immunoblots/western
A
electrophoresis of proteins, blot proteins from gel to a membrane. detect protein by using antibody/antiserum to target the protein.
-a labeled secondary antibody (conj/affinity) is used to bind the first antibody for visualization/to inc the signal.
-primary antibody binds to one of the bands, can be visualized by using a second antibody.
17
Q
Immunoblots/western process
A
-SDS PAGE (protein gel) on proteins
-blot proteins, gel –> membrane
-bind primary antibody
-bind labeled secondary antibody
-detect label
18
Q
Modern DNA sequencing is based on the ______ method
A
Sanger
19
Q
Modern DNA sequencing uses
A
dideoxy nucleotides to stop DNA synthesis by not allowing the 3’ hydroxyl to be used. this creates a series of DNA fragments, size measured by gel electro. the order of size will tell the base sequence.
20
Q
In DNA sequencing, the last base in each fragment is known as the ________
A
dideoxy nucleotide that was used to stop the rxn
21
Q
Sanger DNA sequencing steps
A
-primer extension rxn, replication w ddTTP
-the products of the 4 rxns are: ddA, ddC, ddG, ddT
-electrophoresis of products.
22
Q
Automated DNA sequencing
A
-uses nucleotides tagged w different florescent molecules, products of each nucleotide produces a different color.
-4 reactions completed, then mixed together and run on one lane of gel.
23
Q
Automated DNA sequencing steps
A
-primer extension reactions
-electrophoresis: florescent light emitted by band, laser light points to it, detector of light goes to computer
-laser and detector produces the multicolor graph
24
Q
Northern blots
A
Done to find whether a gene is expressed or not. The other methods detected the presence of a gene. This blot gets RNA from different tissues, runs RNA on agarose gel/blot to membrane, hybridize w comp labeled cDNA probe. this is quantified using densitometer.
25
Gel mobility shift
-this is an assay.
-uses radioactive tracers
-DNA moves through a gel faster, when not bound to protein
-this assay detects interaction between a protein and DNA by reduction of electrophoretic mobility of a small DNA bound to a protein.
26
Gel mobility super shift
the gap between DNA bound to 2 proteins and a DNA protein complex.
27
RNA Polymerase structure
Leads to RNA transcription
-SDS PAGE of RNA polymerase from E coli:
-has these subunits: sigma (70), alpha (40), and omega (10).
-alpha has 2 copies in the holoenzyme
-omega plays a role in enzyme assembly
28
Holoenzyme
does synthesis. recognizes promoter because of the sigma. without the sigma, the holoenzyme still worked with both types of DNA.
29
Core enzyme (has no sigma subunit) cannot transcribe viral DNA (t/f)
true. only holoenzyme.
30
Promoters
this is where RNA polymerase binds.
sigma permits recognition of promoters.
transcription that begins at promoters is specific and directed by sigma.
31
How does core RNA polymerase transcribe nicked DNA
nicks and gaps are good sites for RNA polymerase to bind non specifically.
32
Does holoenzyme or core enzyme bind more tightly? (experiment uses nitrocellulose filters)
holoenzyme. core enzyme binding includes nitrocellulose binding to ssDNA, but not dsDNA.
33
closed promoter complex
holoenzyme loosely bound at promoter, bc DNA is in a closed DS form.
34
open promoter complex
holoenzyme that melted a short DNA region at promoter to form an open promoter complex, with polymerase bound tightly to DNA. (after closed promoter)
35
When RNA polymerase holoenzyme bind to DNA...
-it binds loosely at first
-binds first at promoter
-scans along DNA until promoter is found
36
Polymerase- promoter DNA steps:
-scans for promoter, then holoenzyme loosely binds to core promoter (closed)
-RNA polym melts promoter
-forms open promoter, now tightly bound
-bubble created, transcription
37
core promoter elements are ______ of all genes
upstream
38
bacterial promoters have a __ box and a __ box
-10, -35
39
the -10 and -35 promoter boxes are located ...
10bp upstream of transcription, and 35bp upstream
40
-10 box is
TATAAT
41
-35 box is
TTGACA
42
Down mutation
weakens promoter binding, and inc deviation from consensus seq.
43
up mutation
strengthens promoter binding, dec deviation from the consensus seq
44
UP element
upstream promoter.
-alpha factor binds here.
-upstream of the -10 and -35 boxes, and core promoter.
-increases transcrip activity
45
core promoter element is bound by the ____ factor
sigma
46
Fis sites
rrnB P1 promoter for rrn genes (rRNA genes) in e coli.
-rRNA can quickly turn on fis sites to adapt.
-e coli has 7 rrn genes
-theres 3 enhancers (the fis sites), bound by transcription activator protein: FIS
47
Transcription Initiation needs ______ but isnt ______
template, primer dependent
48
Transcription initiation process
-forms closed promoter
-forms open promoter (melts promoter)
-polymerase at the promoter polymerizes early nucleotides
-transcription becomes long enough to form a stable hybrid w/ template, promoter is cleared.
-now elongation can start.
49
During initiation ___ factor can be recycled to use again with a new core polymerase
sigma
50
Reuse of sigma
core enzyme can release sigma, and sigma can then associate w another core enzyme.
51
Elongation depends on dissociation of the ______
sigma factor
52
Sigma factors have 4 similar regions inside. These 4 sequences are involved in ______
binding to core and DNA
53
Sigma regions 2 and 4
these can interact w core promoter elements.
-10 box can interact w sigma region 2.4
-35 box can interact w sigma region 4.2
54
RNA polymerase can recognize an UP element (t/f)
true
55
Which factor of RNA polymerase can recognize UP elements?
alpha factor of core polymerase.
-the amino of alpha unit will interact with the beta, and alpha will bind to beta.
-carboxyl can interact w UP.
56
The C terminal domain of alpha unit helps the sigma factor of RNA polym bind to core promoter (t/f)
false
57
When binding a promoter with an UP element using sigma factor and the alpha factors C terminal domain:
-theres very strong interaction between polymerase and promoter
-high level of transcription
-alpha helps assembly of RNA polym.
58
Beta and Beta'....
-bind to transcription start site
-recognize promoter
-elongates
59
Sigma factor is only needed during (elongation or initiation)
initiation
60
Core polymerase contains ______
RNA synthesizing machinery
61
Phosphodiester bond formation and DNA binding involve....
Beta and Beta' subunits
62
Major role of alpha subunit
Assembly of core polymerase
63
Beta subunit is located...
near the active site of the RNA polymerase
64
Where are the phosphodiester bonds formed, linking nucleotides?
active site of RNA polymerase
65
The catalytic site of RNA polymerase is located...
inside of Beta'
66
Which of the following is a target of the Cro protein?
OR1
67
When the phage lambda genome is integrated into the bacterial genome, this is referred to as what?
Prophage
68
Which of the following sigma specificity factors is involved in middle gene transcription during SPO1 phage infection of bacteria?
gp28
69
cAMP level is increased by the presence of high levels of glucose. (t/f)
false
70
Which of the following is not used in a Sanger chain termination reaction?
ddUTP
71
Which of the following statement is NOT TRUE regarding the sigma-factor?
It does not have a DNA-binding domain
72
You have performed an in vitro transcription-assay using a cell extract that has mutant C-terminal domain of an alpha subunit. Which of the following would most likely occur?
Polymerase complex would be loosely associated with the promoter
73
Which of the following statement is false regarding the sigma factor?
It does not have a DNA-binding domain.
74
TATAAT is a consensus sequence located at -10 upstream of the transcription start site. (t/f)
True
75
Hydrogen bonds are formed between the amino acids in the repressor and the bases in the DNA. (t/f)
true
76
Transcription in prokaryotes is initiated by sigma factors. (t/f)
true
77
Which of the following is an allosteric protein?
repressor
78
The trp operon consists of ________ genes that encode tryptophan biosynthesis enzymes.
5
79
Lac operon contains
3 structural genes, 1 operator, and 1 promoter are present
80
Beta-galactosidase breaks lactose into __________ and ____________.
glucose, galactose
81
Western blotting detects ___, northern blotting detects ____, southern blotting detects _____, gel mobility shift assay detects ____
protein, RNA, DNA, protein-DNA interaction
82
enhancers...
bind protein factors and stimulate transcription
83
Amino acids whose side chains can interact with the DNA or RNA nucleotide bases via H-bonding include the following:
Asn and Gln
84
Which of the following techniques can be used to study the interaction between repressors and operators?
x-ray diffraction
85
Enhancers are usually quite a distance away from promoters. Which of the following best explains the interaction of such enhancers with promoters?
Promoter and enhancer regions are brought closer by DNA looping.
86
Elongation of transcription involves
polymerization of nucleotides as RNA polym travels along template DNA
87
DNA must _____ ahead of advancing polymerase and ______ behind it
unwind, rewind
88
The unwinding and rewinding of DNA creates tension ahead of the bubble...
which is relieved by topoisomerases.
89
RNA polym. can pause and backtrack during elongation (t/f)
true
90
Pausing of RNA polym. during elongation allows...
allows ribosomes to keep pace with RNA polymerase, and its the first step in termination.
91
Backtracking of RNA polym....
aids in proofreading by extruding the 3' end of the RNA out of the polymerase. If wrong nucleotide is used, that part of the 3' will be cleared.
92
When the polymerase reaches a terminator at the end of a gene...
it falls off the template and releases the RNA
93
2 Main Types of Terminators:
-Intrinsic: function w rho independent RNA polymerase by itself w/o help from other proteins
-rho/p dependent terminators: depend on rho protein.
*rho is hexomer, with ATP activity.
94
intrinsic termination
-inverted repeats: followed by t rich region
-T rich region: non-template strand of a gene. pulls structure out of DNA helix. force of attraction between transcription.
95
Inverted repeats and hair pins
inverted repeats have a transcript to form a hair pin structure bc of complementary base pairing between its sequences, it is self complementary.
96
Bacterial terminators process:
-base pair something to the transcript to destablize RNA/DNA hybrid, causing hairpin
-causes pause of transcript
-a string of U's downstream of hairpin destabilize hybrid and RNA falls of DNA template
-bonds between U and A are weak
97
Rho termination process
rho depression of ability of RNA polym to transcribe phage DNAs in vitro.
-depression is due to termination of transcription
-after termination, polymerase has to reinitiate to begin transcribing again
98
Rho hexamers bind to transcript at...
-rho loading site, the C rich region in transcript
-this activates ATPase, supplying energy to propel along RNA to follow RNA polym
-this chase goes on until RNA polym stalls at terminator site just after making the RNA hairpin
99
Rho process summary
-binding of rho to growing transcript, rho follows polym.
-rho catches polym as it pauses at hair pin
-rho releases transcript from DNA polym complex by unwinding RNA-DNA hybrid
100
Rho protein has no string of T's, only _____
inverted repeat
101
Control of gene expression
to maintain genes Some genes are on and some off, depending on demand
102
E coli cells are grown in both ______
glucose and lactose
103
E coli bacteria prefers ______
glucose, cells will grow fast till exhaustion, then growth stops, for this 1 hr lag period the cells will induce enzymes to metabolize lactose- the lac operon, and growth will resume
104
The lac operon is compose of these three genes that code for E coli, permitting bacteria to use lactose
-galactose permease (lacY)- transports lactose to cells
-B galactosidase (laz Z)- cuts lactose into galactose and glucose
-Galactoside transacetylase (lac A)- unclear function
105
Genes of the Lac Operon are ____
polycistronic.
-the 3 genes are transcribed together, to produce 1 mRNA: a polycistronic message from a single promoter.
106
Each cistron/gene has its own:
RIbosome binding site
107
Each cistron can be translated by separate ribosomes
that bind independently of each other
108
The lac operon will be 'OFF' if theres no _______
lactose
109
When the lac repressor binds to the operator....
-operon is repressed
-prevents RNA polym from binding to promoter
110
Lac operon inducer made from lactose....
binds to repressor protein and changes shape
111
Lac Operon repressor is an _______ protein
allosteric
112
The repressor is no longer bound to lac operon after
shape change
113
Allosteric definition
-binding of 1 molecule to the protein changes shape of a remote site on that protein, altering its interaction with further molecules.
114
When the lac inducer binds to the repressor...
the repressor changes conformation, now favoring release from operator
115
Allolactose forms because
Lac operon is leaky
116
The inducer ______ is an alt form of lactose
allolactose
117
Lac Operators
3 lac operators.
O1, major lac op, is next to promoter.
O2 and O3: aux ops. one is downstream, one is upstream.
all three ops are required for optimum repression.
118
When glucose is present, lac operon is ....
inactive
119
Catabolite
glucose breakdown product. selects in favor of glucose.
120
Catabolite repression
uses breakdown product to repress operon
121
CAP is made from .....
catabolite
122
When glucose decreases, _______ increases
Cyclic AMP
123
CAP binding sites in lac, gal, and ara operons show sequence______
TGTGA
124
Positive control of lac operon
-concentration of cAMP rises as glucose drops
-lack of glucose responds by activating lac promoter
125
Addiyion of _____ leads to activation of lac gene, even in presence of glucose
cAMP
126
Binding of ____ and _____ to activator site helps RNA polym form an open promoter complex
cAMP, CAP
127
Positive controller of lac operon:
cAMP and CAP
128
Lactose is a _______ Tryptophan is a __________
disaccharide, amino acid
129
The CAP-cAMP _____ binds to its target site on DNA
dimer
130
cAMP-CAP activation of lac transcription
-cap/camp dimer bind to DNA
-alpha CTD of RNA polym interacts with CAP
-binding is strengthened between promoter and polymerase
131
trp operon
-e coli trp operon contains genes to make tryptophan
-trp operon codes for anabolic enzymes, to build up a substance
-anabolic enzymes are turned off by a high level of substance
-trp operon is subject to negative control by a repressor when trp levels are elevated, and trp operon starts attenuation at same time
132
When trp is high there is _____ when trp is low there is ______
feedback inhibition, turning on of trp operon
133
_____ genes code for polypeptides in enzymes of trp synthesis
five
134
the trp operator is inside of ______
trp promoter
135
high tryptophan concentration is the signal to...
turn off the operon. trp helps trp repressor bind to trp operator.
136
When theres no tryptophan...
trp repressor doesnt exist, only aporepressor. if aporepressor binds to trp, theres high affinity for trp operator.
137
trp repressor is made of
aporepressor + tryptophan
138
Tryptophan is a .....
corepressor
139
Active repressor is made up of
aporepressor + corepressor (trp)
140
repression of trp operon is ______than lac operon
weaker
141
control of trp operon by Attenuation
-attenuation allows 10 fold control of operons activity
-the 2 loci: trp leader and trp attenuator are in between attenuator and 1st gene
-these 2 loci attenuate or weaken transcript of operon when trp is high
142
TRP Attenuation 2 loci
trp leader: translation start site, 2 trp codons in a row
trp attenuator: transcript termination sequences
143
Trp Attenuation, low v. high trp
low trp: transcription of trp genes, RNA will transcribe through
high trp: 2 hairpins form, attenuiation, premature termination
144
Attenuation process
-causes premature termination of operon transcript when product is abundant
-in low trp, structural genes are transcribed, RNA polym reads thru attenuator
-in high trp, premature termination of transcript.
-stalling only happens when theres starvation of trp, low trp.
145
Attenuator features
-4 inverted repeat elements
-inverted repeats make hairpin structures, theres a hairpin for high trp and low trp
-hairpin followed by string of U's destabilizes binding between transcript and DNA
-theres no stalling of ribosome, ribosome will bind and fall off
146
Defeating attenuation
if AA supply is low, ribosomes stall at tryp codons in the trp leader. trp leader being made as stalling occurs, stalled ribosome influences the way RNA folds. prevents hair pin between elements 1 and 2.
147
Attenuation operates in e coli trp operon if ...
tryptophan is plentiful
148
Overriding attenuation
ribosome translates, hits termination and falls off, allows formation of 2 hair pins, RNA polym falls off
149
Three mechanisms of shifts in bacterial transcription
sigma factor switching
RNA polym switching
antitermination
150
Sigma factor switching
phage infection of bacterium overrides host transcription, and creates a time dependent program of transcription.
change in genes transcribed is caused by a change in transcription machinery in RNA polymerase itself.
151
Sigma factor switching, temporal transcriptopn
-early phage genes transcribed, followed by later genes
-late in infectious cycle theres no transcription of host genes, only phage genes.
152
______ is the key factor in determining specificity of T4 DNA transcription
sigma
153
B subtilis
-phage is SPO1
-used for T4 phage infection
-SPO1 has temporal program of transcription
154
SPO1 temporal transcription
-first 5 min: express early genes
-after 5-10 min: expression of middle genes
-after 10 min-end: late genes expressed
-this relies on host polym binding to early promoter.
-gp28 is an early protein/sigma able to bind to cro RNA polym to make whole RNA polym
155
SPO1 temporal transcription middle proteins
gp33 and gp34 join to become sigma factor
156
transcription switching
-directed by a set of phage encoded sigma factors, that associate w the host core RNA polym.
-sigma factors change the host polym specificity of promoter recognition from early, to middle, to late.
-Host sigma factor is specfic for phage early genes. phage gp28 switches spec to middle, gp33 and gp34 switch spec to late.
157
RNA polymerase encoded in Phage T7
-Phage T7-small genome, fewer genes than SPO1
-three phases of transcript: class 1,2, and 3
-gene 1 is necessary for class 2 and class 3 gene expression
-if gene 1 is mutated, only class 1 is transcribed.
-gene 1 codes for phage specific RNA polym that transcribes T7
158
Host polymerase transcribes the class _ genes
1
159
Class 1 genes, Gene 1
phage polymerase, that can transcribe class 2 and 3 genes.
160
T7 has _ enzyme and _ polymerase
1, 1
161
Class 1 proteins make own
polymerase
162
Virulent phage
replicate and kill host by lysing/breaking open
163
temperate phage
infect but dont kill cells.
has lytic and lysogenic cycles
164
lytic mode
infection goes on as in a virulent phage
-enter, replicate, burst
165
lysogenic mode
phage DNA is integrated into host genome
-enter, manipulate, lyse or integrate
-repressor c1 binds to 2 phage operator bc it can be leaky, this will shut down transcription of all genes except c1
-integrated DNA is prophage
-the phage DNA in lysogen replicates along w host DNA
166
Bacterium harboring integrated phage DNA
lysogens
167
C1, the lysogenic repressor, ...
is supported by C2 and C3
168
Lytic reproduction cycle 3 phases
immediate early, delayed early, late.
genes of these phases are arranged sequentially on the phage DNA
-if repressor overcomes cro protein, lysogenic process occurs
169
DNA exists in the ______ form in a phage
linear
170
after infection of host begins the phage DNA _______
circularizes
171
Phage gene transcription is controlled by
transcriptional switches
172
Antitermination
-transcriptional switch used by phage
-host RNA polym transcribes early genes 2st
-gene product serves as antiterminator that permits RNA polymerase to ignore terminators at the end of the immediate early genes
-same promoters are used for early and middle transcript
-late genes transcribed when another antiterminator permits transcript of late genes from late promoter to continue w/o premature termination
173
Immediate early genes
Cro- repressor of C1, allows lytic cycle to continue
N- antiterminator, prevents rna polym from terminating, allows it to continue
Q is a middle antiterminator to continue transcript thru late genes
174
genetic sites surrounding N gene
Left promoter PL, Operator OL, terminator
175
When N is present
N binds transcript of N utilization site, interacts w protein complex bound to polym. Polym ignores terminator, continues to middle genes
176
Antitermination in the late region requires
Q
177
Q binds to ....
Q binding region of qut-site, as RNA polym is stalled downstream of late promoter.
-binding of q to polym alters the enzyme so it can ignore terminator
178
Phage establishes lysogeny by ...
-producing repressor, to bind to early operators, if it binds RNA polym cant go over these sites
-middle gene products used
-integration to host genome
-c2 and c3 allows transcript of c1 and repressor, by grabbing RNA polym to C1
-promoter used to establish lysogeny is PRE
179
Lysogeny operator sites over lap with...
PR and PL sites
180
Middle transcript from PR produces...
C2 mRNA translated to C2 protein
181
C2 alows RNA polym to bind to ....
PRE and transcribe C1 gene, result is repressor transcription
182
Autoregulation of C1 gene during Lysogeny
when repressor binds as dimer to operator, repressor turns off further early transcription, interrupts lytic cycle, stimulates own synthesis by activating PRM, and turn off of cro.
Cro must be turned off bc it can counter repressor activity.
183
PRM is only for going over the
C1 gene.
184
Lysogeny repressor has affinity for
Br1 and Br2
185
Cro is able to bind to the OR's
has affinity for Or3
186
Repressor protein
dimer of 2 identical subunits:
-amino terminal- DNA binding end
-carboxyl terminal- repressor-repressor interaction, and allows dimerization
187
Repressor Binds to __ and __ but leaves __
Or1, Or2, Or3
188
RNA polymerase binds to PRM which overlaps with
Or3, causing it to contacts the repressor bound to Or2.
189
Protein protein interaction is required for
promoter to work
190
High levels of repressor can...
repress transcription from PRM
191
Repressor dimers are bound to OL1, OL2, and OL3 via
DNA looping
192
Balance between lysis/lysogeny is
delicate
193
If lytic infection occurs in experiment
progeny spreads to infect other cells, circular hole seen is called plaque
194
infection 100%lytic gives
clear plaque
195
Lysogen plaques are usually turbid meaning....
live lysogen is present
196
Repressor blocking OR1, OR2, OL1, and OL2
turns off early transcription, leads to lysogeny
197
Cro blocks OR3 and OL3, turning off transcription
leads to lytic infection
198
If c1 wins, theres ____ if cro wins theres ____
lysogeny, lytic
199
When lysogen suffers DNA damage
event is seen in coprotease activity in RecA repair protein. Repressors are caused to cut in half, removing them from operators. Lytic cycle is induced, progeny phage can escape damage in host. RecA modifies C1/repressor.
200
Repressors have recognition helices that lie in the ______ of the operator. ________ of this binding depends on Amino acids in the recognition helices.
major groove, amino acids
201
Repressor-DNA interaction site
recognition helices of repressors fit sideways into major groove of op DNA. some AA's on DNA side of recognition helix make specific contact w bases in the operator, determining specificity of protein DNA interactions. changing AA's can change specificity of repressor.
202
Probing binding specificity by mutagenesis
key AA's in the recognition helices of 2 repressors, these AA's are different between the 2 repressors.
203
The lambda repressor has an extra ____
motif. an amino terminal arm that binds by embracing DNA. Cro and lambda repressor share affinity for the same operators, but especially OR1 and OR3.
204
Lambda repressor-operator structure:
-recognition helices of each repressor are in the DNA major grooves in the 2 half sites
-helices approach e/o to hold the repressors together in the repressor dimer
-bending of DNA in the two ends of the DNA fragment as it curves around the repressor dimer
205
There are ______ bonds between lambda repressor and base pairs in the major groove
hydrogen
206
AA- DNA backbone interactions
H bond at Gln33 maximizes electrostatic attraction between pos charged amino end of alpha helix and neg charged DNA
-attraction works to stabilize the bond
207
X ray crystallography of repressor/operator complex shows...
H bonding at 3 Gln residues in recognition helix to 3 bp's in operator
-van der waals contact between one of these glutamines and base in the operator
208
analysis of phage 434 repressor operator complex shows...
DNA deviates from its normal shape
DNA bends to accomodate base/AA contacts
central part of helix is wound tightly
209
specificity of binding between a protein and a specific stretch of DNA relates to:
-interactions between bases/AA's
-ability of DNA to assume a certain shape, relating to DNAs base seq.
210
H bonding of base pairs
four base pairs= four different H bonding profiles to AA's going to the major or minor groove.
211
target sites for DNA binding proteins are
symmetric or repeated
212
most DNA binding proteins are....
dimers, that enhance binding between DNA and protein
213
multimeric DNA binding proteins have a higher affinity for binding sites on DNA than...
multiple monomeric proteins that bind independently
214
DNA binding sites can influence interactions at remote sites in DNA, with enhancers occuring
2000-3000 upstream of gene
215
enhancers
-can bind to general transcription factors
-exert function from distance
-non promoter DNA elements that bind to protein factors and stim transcription
-enhancers interact w promoter by DNA looping
