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molecular ex 2 Flashcards

1
Q

Labeled tracers are…

A

radioactive.
ph32 used to detect DNA,
sulfur35 to detect proteins.

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2
Q

autoradiography

A

detects radioactive compounds using photographic emulsion (x ray), specifically tracers and labels

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3
Q

nonradioactive tracers

A

use enzymes instead of radioactivity, uses multiplier effect of an enzyme to generate a signal

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4
Q

Nonradioactive tracers may use _____ for dUTP along with avidin because it has a natural affinity for avidin

A

Biotin

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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

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6
Q

Nucleic acid hybridization

A

one single stranded nucleic acid forms a double helix with another single strand of complementary base sequence

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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.

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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.

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9
Q

southern blot bands are seen with

A

x ray film or autoradiography

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10
Q

southern blot bands could show

A

multiple genes or several restriction sites in gene

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11
Q

__________ can be used in forensic labs to detect people using DNA

A

southern blots

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12
Q

mini satellite DNA

A

sequence of bases unique to every person repeated several times, called DNA fingerprint (not used anymore)

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13
Q

Parts of DNA fingerprint pattern can be inherited (Mendelian) (t/f)

A

True

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14
Q

DNA fingerprinting uses

A

set of probes to give set of simple patterns, and uses probes for just a single locus

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15
Q

Immunoblots AKA Western blots are similar to

A

southern blots

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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.

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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

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18
Q

Modern DNA sequencing is based on the ______ method

A

Sanger

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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.

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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

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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.

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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.

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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

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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.

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25
Q

Gel mobility shift

A

-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.

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26
Q

Gel mobility super shift

A

the gap between DNA bound to 2 proteins and a DNA protein complex.

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27
Q

RNA Polymerase structure

A

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

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28
Q

Holoenzyme

A

does synthesis. recognizes promoter because of the sigma. without the sigma, the holoenzyme still worked with both types of DNA.

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29
Q

Core enzyme (has no sigma subunit) cannot transcribe viral DNA (t/f)

A

true. only holoenzyme.

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30
Q

Promoters

A

this is where RNA polymerase binds.
sigma permits recognition of promoters.
transcription that begins at promoters is specific and directed by sigma.

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31
Q

How does core RNA polymerase transcribe nicked DNA

A

nicks and gaps are good sites for RNA polymerase to bind non specifically.

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32
Q

Does holoenzyme or core enzyme bind more tightly? (experiment uses nitrocellulose filters)

A

holoenzyme. core enzyme binding includes nitrocellulose binding to ssDNA, but not dsDNA.

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33
Q

closed promoter complex

A

holoenzyme loosely bound at promoter, bc DNA is in a closed DS form.

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34
Q

open promoter complex

A

holoenzyme that melted a short DNA region at promoter to form an open promoter complex, with polymerase bound tightly to DNA. (after closed promoter)

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35
Q

When RNA polymerase holoenzyme bind to DNA…

A

-it binds loosely at first
-binds first at promoter
-scans along DNA until promoter is found

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36
Q

Polymerase- promoter DNA steps:

A

-scans for promoter, then holoenzyme loosely binds to core promoter (closed)
-RNA polym melts promoter
-forms open promoter, now tightly bound
-bubble created, transcription

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37
Q

core promoter elements are ______ of all genes

A

upstream

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38
Q

bacterial promoters have a __ box and a __ box

A

-10, -35

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39
Q

the -10 and -35 promoter boxes are located …

A

10bp upstream of transcription, and 35bp upstream

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40
Q

-10 box is

A

TATAAT

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41
Q

-35 box is

A

TTGACA

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42
Q

Down mutation

A

weakens promoter binding, and inc deviation from consensus seq.

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43
Q

up mutation

A

strengthens promoter binding, dec deviation from the consensus seq

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44
Q

UP element

A

upstream promoter.
-alpha factor binds here.
-upstream of the -10 and -35 boxes, and core promoter.
-increases transcrip activity

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45
Q

core promoter element is bound by the ____ factor

A

sigma

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46
Q

Fis sites

A

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

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47
Q

Transcription Initiation needs ______ but isnt ______

A

template, primer dependent

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48
Q

Transcription initiation process

A

-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.

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49
Q

During initiation ___ factor can be recycled to use again with a new core polymerase

A

sigma

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50
Q

Reuse of sigma

A

core enzyme can release sigma, and sigma can then associate w another core enzyme.

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51
Q

Elongation depends on dissociation of the ______

A

sigma factor

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52
Q

Sigma factors have 4 similar regions inside. These 4 sequences are involved in ______

A

binding to core and DNA

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53
Q

Sigma regions 2 and 4

A

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

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54
Q

RNA polymerase can recognize an UP element (t/f)

A

true

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55
Q

Which factor of RNA polymerase can recognize UP elements?

A

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.

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56
Q

The C terminal domain of alpha unit helps the sigma factor of RNA polym bind to core promoter (t/f)

A

false

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57
Q

When binding a promoter with an UP element using sigma factor and the alpha factors C terminal domain:

A

-theres very strong interaction between polymerase and promoter
-high level of transcription
-alpha helps assembly of RNA polym.

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58
Q

Beta and Beta’….

A

-bind to transcription start site
-recognize promoter
-elongates

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59
Q

Sigma factor is only needed during (elongation or initiation)

A

initiation

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60
Q

Core polymerase contains ______

A

RNA synthesizing machinery

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61
Q

Phosphodiester bond formation and DNA binding involve….

A

Beta and Beta’ subunits

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62
Q

Major role of alpha subunit

A

Assembly of core polymerase

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63
Q

Beta subunit is located…

A

near the active site of the RNA polymerase

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64
Q

Where are the phosphodiester bonds formed, linking nucleotides?

A

active site of RNA polymerase

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65
Q

The catalytic site of RNA polymerase is located…

A

inside of Beta’

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66
Q

Which of the following is a target of the Cro protein?

A

OR1

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67
Q

When the phage lambda genome is integrated into the bacterial genome, this is referred to as what?

A

Prophage

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68
Q

Which of the following sigma specificity factors is involved in middle gene transcription during SPO1 phage infection of bacteria?

A

gp28

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69
Q

cAMP level is increased by the presence of high levels of glucose. (t/f)

A

false

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70
Q

Which of the following is not used in a Sanger chain termination reaction?

A

ddUTP

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71
Q

Which of the following statement is NOT TRUE regarding the sigma-factor?

A

It does not have a DNA-binding domain

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72
Q

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?

A

Polymerase complex would be loosely associated with the promoter

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73
Q

Which of the following statement is false regarding the sigma factor?

A

It does not have a DNA-binding domain.

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74
Q

TATAAT is a consensus sequence located at -10 upstream of the transcription start site. (t/f)

A

True

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75
Q

Hydrogen bonds are formed between the amino acids in the repressor and the bases in the DNA. (t/f)

A

true

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76
Q

Transcription in prokaryotes is initiated by sigma factors. (t/f)

A

true

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77
Q

Which of the following is an allosteric protein?

A

repressor

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78
Q

The trp operon consists of ________ genes that encode tryptophan biosynthesis enzymes.

A

5

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79
Q

Lac operon contains

A

3 structural genes, 1 operator, and 1 promoter are present

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80
Q

Beta-galactosidase breaks lactose into __________ and ____________.

A

glucose, galactose

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81
Q

Western blotting detects ___, northern blotting detects ____, southern blotting detects _____, gel mobility shift assay detects ____

A

protein, RNA, DNA, protein-DNA interaction

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82
Q

enhancers…

A

bind protein factors and stimulate transcription

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83
Q

Amino acids whose side chains can interact with the DNA or RNA nucleotide bases via H-bonding include the following:

A

Asn and Gln

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84
Q

Which of the following techniques can be used to study the interaction between repressors and operators?

A

x-ray diffraction

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85
Q

Enhancers are usually quite a distance away from promoters. Which of the following best explains the interaction of such enhancers with promoters?

A

Promoter and enhancer regions are brought closer by DNA looping.

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86
Q

Elongation of transcription involves

A

polymerization of nucleotides as RNA polym travels along template DNA

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87
Q

DNA must _____ ahead of advancing polymerase and ______ behind it

A

unwind, rewind

88
Q

The unwinding and rewinding of DNA creates tension ahead of the bubble…

A

which is relieved by topoisomerases.

89
Q

RNA polym. can pause and backtrack during elongation (t/f)

A

true

90
Q

Pausing of RNA polym. during elongation allows…

A

allows ribosomes to keep pace with RNA polymerase, and its the first step in termination.

91
Q

Backtracking of RNA polym….

A

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
Q

When the polymerase reaches a terminator at the end of a gene…

A

it falls off the template and releases the RNA

93
Q

2 Main Types of Terminators:

A

-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
Q

intrinsic termination

A

-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
Q

Inverted repeats and hair pins

A

inverted repeats have a transcript to form a hair pin structure bc of complementary base pairing between its sequences, it is self complementary.

96
Q

Bacterial terminators process:

A

-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
Q

Rho termination process

A

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
Q

Rho hexamers bind to transcript at…

A

-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
Q

Rho process summary

A

-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
Q

Rho protein has no string of T’s, only _____

A

inverted repeat

101
Q

Control of gene expression

A

to maintain genes Some genes are on and some off, depending on demand

102
Q

E coli cells are grown in both ______

A

glucose and lactose

103
Q

E coli bacteria prefers ______

A

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
Q

The lac operon is compose of these three genes that code for E coli, permitting bacteria to use lactose

A

-galactose permease (lacY)- transports lactose to cells
-B galactosidase (laz Z)- cuts lactose into galactose and glucose
-Galactoside transacetylase (lac A)- unclear function

105
Q

Genes of the Lac Operon are ____

A

polycistronic.

-the 3 genes are transcribed together, to produce 1 mRNA: a polycistronic message from a single promoter.

106
Q

Each cistron/gene has its own:

A

RIbosome binding site

107
Q

Each cistron can be translated by separate ribosomes

A

that bind independently of each other

108
Q

The lac operon will be ‘OFF’ if theres no _______

A

lactose

109
Q

When the lac repressor binds to the operator….

A

-operon is repressed
-prevents RNA polym from binding to promoter

110
Q

Lac operon inducer made from lactose….

A

binds to repressor protein and changes shape

111
Q

Lac Operon repressor is an _______ protein

A

allosteric

112
Q

The repressor is no longer bound to lac operon after

A

shape change

113
Q

Allosteric definition

A

-binding of 1 molecule to the protein changes shape of a remote site on that protein, altering its interaction with further molecules.

114
Q

When the lac inducer binds to the repressor…

A

the repressor changes conformation, now favoring release from operator

115
Q

Allolactose forms because

A

Lac operon is leaky

116
Q

The inducer ______ is an alt form of lactose

A

allolactose

117
Q

Lac Operators

A

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
Q

When glucose is present, lac operon is ….

A

inactive

119
Q

Catabolite

A

glucose breakdown product. selects in favor of glucose.

120
Q

Catabolite repression

A

uses breakdown product to repress operon

121
Q

CAP is made from …..

A

catabolite

122
Q

When glucose decreases, _______ increases

A

Cyclic AMP

123
Q

CAP binding sites in lac, gal, and ara operons show sequence______

A

TGTGA

124
Q

Positive control of lac operon

A

-concentration of cAMP rises as glucose drops
-lack of glucose responds by activating lac promoter

125
Q

Addiyion of _____ leads to activation of lac gene, even in presence of glucose

A

cAMP

126
Q

Binding of ____ and _____ to activator site helps RNA polym form an open promoter complex

A

cAMP, CAP

127
Q

Positive controller of lac operon:

A

cAMP and CAP

128
Q

Lactose is a _______ Tryptophan is a __________

A

disaccharide, amino acid

129
Q

The CAP-cAMP _____ binds to its target site on DNA

A

dimer

130
Q

cAMP-CAP activation of lac transcription

A

-cap/camp dimer bind to DNA
-alpha CTD of RNA polym interacts with CAP
-binding is strengthened between promoter and polymerase

131
Q

trp operon

A

-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
Q

When trp is high there is _____ when trp is low there is ______

A

feedback inhibition, turning on of trp operon

133
Q

_____ genes code for polypeptides in enzymes of trp synthesis

A

five

134
Q

the trp operator is inside of ______

A

trp promoter

135
Q

high tryptophan concentration is the signal to…

A

turn off the operon. trp helps trp repressor bind to trp operator.

136
Q

When theres no tryptophan…

A

trp repressor doesnt exist, only aporepressor. if aporepressor binds to trp, theres high affinity for trp operator.

137
Q

trp repressor is made of

A

aporepressor + tryptophan

138
Q

Tryptophan is a …..

A

corepressor

139
Q

Active repressor is made up of

A

aporepressor + corepressor (trp)

140
Q

repression of trp operon is ______than lac operon

A

weaker

141
Q

control of trp operon by Attenuation

A

-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
Q

TRP Attenuation 2 loci

A

trp leader: translation start site, 2 trp codons in a row
trp attenuator: transcript termination sequences

143
Q

Trp Attenuation, low v. high trp

A

low trp: transcription of trp genes, RNA will transcribe through
high trp: 2 hairpins form, attenuiation, premature termination

144
Q

Attenuation process

A

-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
Q

Attenuator features

A

-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
Q

Defeating attenuation

A

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
Q

Attenuation operates in e coli trp operon if …

A

tryptophan is plentiful

148
Q

Overriding attenuation

A

ribosome translates, hits termination and falls off, allows formation of 2 hair pins, RNA polym falls off

149
Q

Three mechanisms of shifts in bacterial transcription

A

sigma factor switching
RNA polym switching
antitermination

150
Q

Sigma factor switching

A

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
Q

Sigma factor switching, temporal transcriptopn

A

-early phage genes transcribed, followed by later genes
-late in infectious cycle theres no transcription of host genes, only phage genes.

152
Q

______ is the key factor in determining specificity of T4 DNA transcription

A

sigma

153
Q

B subtilis

A

-phage is SPO1
-used for T4 phage infection
-SPO1 has temporal program of transcription

154
Q

SPO1 temporal transcription

A

-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
Q

SPO1 temporal transcription middle proteins

A

gp33 and gp34 join to become sigma factor

156
Q

transcription switching

A

-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
Q

RNA polymerase encoded in Phage T7

A

-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
Q

Host polymerase transcribes the class _ genes

A

1

159
Q

Class 1 genes, Gene 1

A

phage polymerase, that can transcribe class 2 and 3 genes.

160
Q

T7 has _ enzyme and _ polymerase

A

1, 1

161
Q

Class 1 proteins make own

A

polymerase

162
Q

Virulent phage

A

replicate and kill host by lysing/breaking open

163
Q

temperate phage

A

infect but dont kill cells.
has lytic and lysogenic cycles

164
Q

lytic mode

A

infection goes on as in a virulent phage
-enter, replicate, burst

165
Q

lysogenic mode

A

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
Q

Bacterium harboring integrated phage DNA

A

lysogens

167
Q

C1, the lysogenic repressor, …

A

is supported by C2 and C3

168
Q

Lytic reproduction cycle 3 phases

A

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
Q

DNA exists in the ______ form in a phage

A

linear

170
Q

after infection of host begins the phage DNA _______

A

circularizes

171
Q

Phage gene transcription is controlled by

A

transcriptional switches

172
Q

Antitermination

A

-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
Q

Immediate early genes

A

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
Q

genetic sites surrounding N gene

A

Left promoter PL, Operator OL, terminator

175
Q

When N is present

A

N binds transcript of N utilization site, interacts w protein complex bound to polym. Polym ignores terminator, continues to middle genes

176
Q

Antitermination in the late region requires

A

Q

177
Q

Q binds to ….

A

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
Q

Phage establishes lysogeny by …

A

-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
Q

Lysogeny operator sites over lap with…

A

PR and PL sites

180
Q

Middle transcript from PR produces…

A

C2 mRNA translated to C2 protein

181
Q

C2 alows RNA polym to bind to ….

A

PRE and transcribe C1 gene, result is repressor transcription

182
Q

Autoregulation of C1 gene during Lysogeny

A

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
Q

PRM is only for going over the

A

C1 gene.

184
Q

Lysogeny repressor has affinity for

A

Br1 and Br2

185
Q

Cro is able to bind to the OR’s

A

has affinity for Or3

186
Q

Repressor protein

A

dimer of 2 identical subunits:
-amino terminal- DNA binding end
-carboxyl terminal- repressor-repressor interaction, and allows dimerization

187
Q

Repressor Binds to __ and __ but leaves __

A

Or1, Or2, Or3

188
Q

RNA polymerase binds to PRM which overlaps with

A

Or3, causing it to contacts the repressor bound to Or2.

189
Q

Protein protein interaction is required for

A

promoter to work

190
Q

High levels of repressor can…

A

repress transcription from PRM

191
Q

Repressor dimers are bound to OL1, OL2, and OL3 via

A

DNA looping

192
Q

Balance between lysis/lysogeny is

A

delicate

193
Q

If lytic infection occurs in experiment

A

progeny spreads to infect other cells, circular hole seen is called plaque

194
Q

infection 100%lytic gives

A

clear plaque

195
Q

Lysogen plaques are usually turbid meaning….

A

live lysogen is present

196
Q

Repressor blocking OR1, OR2, OL1, and OL2

A

turns off early transcription, leads to lysogeny

197
Q

Cro blocks OR3 and OL3, turning off transcription

A

leads to lytic infection

198
Q

If c1 wins, theres ____ if cro wins theres ____

A

lysogeny, lytic

199
Q

When lysogen suffers DNA damage

A

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
Q

Repressors have recognition helices that lie in the ______ of the operator. ________ of this binding depends on Amino acids in the recognition helices.

A

major groove, amino acids

201
Q

Repressor-DNA interaction site

A

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
Q

Probing binding specificity by mutagenesis

A

key AA’s in the recognition helices of 2 repressors, these AA’s are different between the 2 repressors.

203
Q

The lambda repressor has an extra ____

A

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
Q

Lambda repressor-operator structure:

A

-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
Q

There are ______ bonds between lambda repressor and base pairs in the major groove

A

hydrogen

206
Q

AA- DNA backbone interactions

A

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
Q

X ray crystallography of repressor/operator complex shows…

A

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
Q

analysis of phage 434 repressor operator complex shows…

A

DNA deviates from its normal shape
DNA bends to accomodate base/AA contacts
central part of helix is wound tightly

209
Q

specificity of binding between a protein and a specific stretch of DNA relates to:

A

-interactions between bases/AA’s
-ability of DNA to assume a certain shape, relating to DNAs base seq.

210
Q

H bonding of base pairs

A

four base pairs= four different H bonding profiles to AA’s going to the major or minor groove.

211
Q

target sites for DNA binding proteins are

A

symmetric or repeated

212
Q

most DNA binding proteins are….

A

dimers, that enhance binding between DNA and protein

213
Q

multimeric DNA binding proteins have a higher affinity for binding sites on DNA than…

A

multiple monomeric proteins that bind independently

214
Q

DNA binding sites can influence interactions at remote sites in DNA, with enhancers occuring

A

2000-3000 upstream of gene

215
Q

enhancers

A

-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

Flash Cards (21 decks)

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