Exam 2

Front 1

Order of greatest to least RNA?

Back 1

rRNA>tRNA>mRNA>fRNA

Front 2

Addition of nucleotide to DNA (draw)

Back 2

Refer to lecture 5.

Front 3

Promoters

Back 3

DNA sequences that guide RNAP to the beginning of a
gene (transcription initiation site).

Front 4

Terminators

Back 4

DNA sequences that specify the termination of RNA
synthesis and release of RNAP from the DNA.

Front 5

Steps of RNAP

Back 5

1) Initiation.
2) Elongation.
3) Termination.

Front 6

Mapping Promoters

Back 6

DNA sequences that guide
RNAP to the start of a gene
(transcription initiation site)

Front 7

Describe the steps of the experiment performed to find where RNAP is on a strand of DNA.

Back 7

Front 8

Describe steps to find location of RNAP on DNA strand.

Back 8

1. Radioactively label DNA fragments at one end of strand.
2. Treat with DNAse (No cuts are made where RNAP is bound)
3. Isolate labelled DNA fragments and denature
4. No cuts are made in the area where RNA polymerase was bound.
5. Isolate labelled DNA fragments and denature. Only labelled fragments are detected in the next step.
6. Separate fragments by gel electrophoresis
7. Missing bands indicate where RNA polymerase was bound to DNA

Front 9

Coding Strand is also known as...

Back 9

Nontemplate strand, Sense strand.

Front 10

Promoters consist of..

Back 10

-35, -10 (Pribnow Box)

Front 11

The closer the match to the -10 and -35 boxes
consensus...

Back 11

the stronger the promoter.

Front 12

Spacers that are longer or shorter than the
consensus length...

Back 12

make weak promoters

Front 13

Holoenzyme

Back 13

2 α, 1 β, 1 β', 1 σ

Front 14

Core enzyme

Back 14

2 α, 1 β, 1 β'

Front 15

Functions of α, β, β', σ?

Back 15

σ recognizes promoter sequences on DNA & is essential for assembly and
for activation of the enzyme; β'
binds DNA; β binds NTPs and interacts with σ

Front 16

σ70, σ32, σ60

Back 16

principal σ, heat shock, nitrogen starvation,

Front 17

σ4 and σ2 bind to which sequences, respectively?

Back 17

-35, -10

Front 18

An asymmetrical promoter

Back 18

defines which direction the
polymerase will initiate
synthesis.

Front 19

Steps of RNAP.

Back 19

1) Binding of RNA polymerase holoenzyme to form a closed complex
2) Formation of an open complex
3) Addition of 6-10 NTPs
4) Dissociation of sigma

Front 20

Trigger loop:

Back 20

A key RNAP loop that recognizes the correct NTP substrate at the
active site of RNA polymerase. It changes its conformation from a disordered
loop to two α helices when the correct NTP enters the active site and triggers
phosphodiester bond formation.

Front 21

NADFDGD motif

Back 21

To discriminate rNTPs from dNTPs, RNAP recognizes the 2’-
OH group of the ribose via hydrogen bonding with the side
chain of an Asn (N) residue found in the universally
conserved NADFDGD motif . Mutation of this Asn
residue increases dNTP incorporation.

Front 22

Mg+ ions of RNAP

Back 22

-Mg+ ions in active site of RNAP are responsible for phosphodiester bond formation.
-1 of Mg+ ions is responsible for keeping incoming phosphates of nucleotide spatially aligned.

Front 23

If RNAP incorporates a mismatched nucleotide..(write down steps)

Back 23

1) Pause elongation
2) Backtrack to form frayed RNA 3' terminal nucleotide.
3) Gre factor or intrinsic cleavage to remove nucleotides
4) RNA hydrolysis makes a new 3' OH group at end of RNA to restart elongation.

Front 24

Rho Dependent Termination

Back 24

Rho factor binds the end of RNA chain and slides along the strang towards the open bubble complex. When the factor catches polymerase, it causes termination of transcription.

Front 25

Rho Independent Termination

Back 25

You should be able to draw stem loop structure first!

-Because the uracils will only weakly pair with the adenines, the RNA chain can easily be released from the DNA template, terminating transcription.

Front 26

Translocation

Back 26

Once the correct nucleotide is added, the TEMPLATE has to be moved such that the next nucleotide is positioned correctly in the active site
of RNAP

New data suggest a role for key structural elements of RNAP in this process

Front 27

Rho

Back 27

ATP-dependent helicase

– it moves along the RNA transcript, finds the "bubble", unwinds it and releases the RNA chain.

Front 28

How does RNAP let go of the promoter and translocate downstream?

Back 28

Synthesis of RNA chains destabilize the interactions between Sigma 4 and the Beta flap (destabilizing interaction between the -10 and -35 elements), which allow RNAP to let go of the promoter.

Front 29

Rut Site

Back 29

Rho initially binds to rut site on mRNA

Front 30

Allosteric Model of Rho Termination

Back 30

Termination induced by nascent RNA that alters the RNAP elongation complex

Front 31

Positive Regulation (activation)

Back 31

a positive regulatory
factor (activator) improves the ability of RNAP to
bind to and initiate transcription at a weak promoter.

Front 32

Negative Regulation (repression)

Back 32

a negative regulatory
factor (repressor) blocks the ability of RNAP to
bind to and initiate transcription at a strong promoter

Front 33

polycistronic mRNA

Back 33

An mRNA that codes for more than one protein.

Front 34

Operons

Back 34

A unit made up of linked genes that is thought to regulate other genes responsible for protein synthesis.

Front 35

Operators / Enhancers

Back 35

-Operators = Repressor Binding Sites

-Enhancers = Activator Binding Sites

Front 36

Constitutive genes

Back 36

are expressed all of the
time. i.e. glucose metabolizing

Front 37

Inducible / Adaptive genes

Back 37

are normally off and are
turned on by the presence of an inducer substance. i.e. lactose metabolizing

Front 38

Repressible genes

Back 38

are normally on and are
turned off by the presence of a repressor substance.

Front 39

repressor protein (LacI)

Back 39

It is an allosteric protein
with two binding sites: either binds Operator DNA or inducer (allolactose).

Front 40

The lac promoter region has two binding sites:

Back 40

1) RNA polymerase binding site

2) CAP-binding site (for catabolite activator protein), an example of positive control

Front 41

What activators work by Allostery rather than Recruitment of RNA polymerase?

Back 41

NtrC, MerR

Front 42

NtrC

Back 42

interacts with its DNA binding site only after phosphorylation by NtrB in response to nitrogen starvation MerR

Front 43

MerR

Back 43

Binds DNA, but Alters its Conformation only in Response to Mercury Binding

Front 44

NusA

Back 44

-elongation factor, promotes termination
-NusA
enables the formation of stable ECs (elongation complexes), which leads to
processive transcription and read-through of termination sites.

Front 45

NusG

Back 45

-is an essential bacterial regulator of the RNAP
-enables transcription:translation coupling, or to ρ factor, to support ρ-dependent
termination.

Front 46

NusB

Back 46

-has single stranded RNA binding activity and forms a stable complex with NusE. This
heterodimer formation enhances the RNA binding affinity of the complex.

Front 47

NusE

Back 47

NusE is involved in translation

Front 48

Eukaryotic Promoter

Back 48

TATA box (analagous to -10 box), CAAT box (-30 in eukaryotes), GC box.

Front 49

Core promoter elements? Proximal promoter elements?

Back 49

Core promoter elements = TATA box; Proximal promoter elements = CAAT + GC box.

Front 50

Deletion of TATA box

Back 50

heterogeneity of transcript start
site without eliminating transcription

Front 51

Distant-independent elements

Back 51

silencers / enhancers

Front 52

Transcription Factors

Back 52

Trans acting factors that alter
the rate of transcription. Often bind to DNA.

Front 53

Transcriptional activators

Back 53

increase rate of transcription

Front 54

Transcriptional repressors

Back 54

decrease the rate of
transcription

Front 55

Common Domains of transcription Factors

Back 55

1) DNA binding domain [binds to DNA sequences]
2) Activation domain [Interacts with basal machinery to
elevate the rate of transcription]
3) Dimerization domain [mediates protein-protein interaction.]
4) Ligand binding domain [the function of some transcription
factors is altered by the binding of a molecule]

Front 56

Basal Transcription Factors

Back 56

-factors required for initiation of
transcription of genes; bind to a core promoter (TATA box, -30)

Front 57

Basal Transcription Factors for RNA Polymerase II

Back 57

TFIID, TFIIA, TFIIB, RNA Polymerase II, TFIIF, TFIIE, TFIIH.

Front 58

TFIID

Back 58

-Consists of TBP (TATA box binding protein) + TAFs (TBP associated factors).
-Bind to core promoter
-Kinks DNA at each end of the TATA box
-First step in basal transcription is binding of TFIID to core promoter
-Other core factors bind to TFIID

Front 59

TFIIA

Back 59

Increase affinity of TBP for DNA in vitro.

Front 60

What are the core factors? (Proteins that bind the promoter)

Back 60

TFIIA, TFIIB

Front 61

Preinitiaton Site (PIC)

Back 61

a large complex of proteins that is necessary for the transcription of protein-coding genes in eukaryotes (+archaea). The preinitiation complex helps position RNA polymerase II over gene transcription start sites, denatures the DNA, and positions the DNA in the RNA polymerase II active site for transcription

Front 62

TFIIH

Back 62

has both helicase
and kinase activities
that can unwind DNA
and phosphorylate
the CTD tail of RNA Pol II

Front 63

YS2PTS5PS7

Back 63

CTD heptapeptide repeats of RNA Polymerase II.

-Phosphorylation of these serine repeats during promoter escape / capping / splicing / poly A addition.

Front 64

How does addition of the polyA tail occur?

Back 64

-RNA polymerase II transcribes well past the end of the gene
-After the end of the gene has been reached, RNA polymerase II passes through an AATAA sequence, which lies beyond the 3' end of the coding sequence.
3. The pre-mRNA, carrying this signal, AAUAA, is then cleaved by a special endonuclease that recognizes the signal and cuts at a site 11 to 30 residues to its 3' side.

Front 65

Poly A Polymerase (PAP)

Back 65

-Poly A polymerase adds A's to the 3' end.
-Binding of poly A binding protein (PABP) enhances the rate of poly a polymerase

Front 66

Why is capping /polyadenylation of mRNA at the 3' end important?

Back 66

-Transport of DNA
-Protects from nucleases
-Stabilizes

Front 67

Rat1 Protein

Back 67

-Protein present on RNA pol II
-Loads onto remainder of transcript and starts chewing 5' --> 3' direction.
-Promotes termination when reaches RNAP , allosterically altering it.

Front 68

Alpha Amantin

Back 68

From the amanita mushroom

Front 69

Actinomysin D

Back 69

Inhibits all RNA polymerases; blocks elongation

Front 70

Rifampicin

Back 70

Blocks promoter clearance / inhibits elongation

Front 71

mRNA processing in Prokaryotes vs. Eukaryotes

Back 71

-Prokaryotes: no significant modification
-Eukaryotes:
a) 5' capping
b) 3' cleavage and polyadenylation
c) removal of introns (nuclear splicing)

Front 72

C Terminal Domain of RNA Polymerase

Back 72

Has serine repeats that are differentially modified as the cell goes through different stages (that recruit different processing factors)

Front 73

Forward Translocation

Back 73

Forward movement about RNAP with respect to RNA/DNA hybrid by 1 or several nucleotides without transcript elongation, thus promoting release of transcript.

Front 74

Roles of Nus G / Nus A in termination

Back 74

Nus G = Rho dependent termination

Nus A = Rho independent termination by helping to form the stem loop structure.

Front 75

IPTG/XGAL

Back 75

Allolactose analogs

Front 76

Pribnow Box

Back 76

A/T rich, ideal for unwinding

Front 77

Sanger method (Chain termination)

Back 77

-ddNTPs will be radioactively or fluorescently labelled
-The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP) which are the chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, thus terminating DNA strand extension and resulting in DNA fragments of varying length.
-newly synthesized and labelled DNA fragments are heat denatured, and separated by size by gel electrophoresis
-the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image
-A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a dideoxynucleotide
-The relative positions can then be used to read a DNA sequence

Front 78

Maxam-Gilbert method of Sequencing

Back 78

-The method requires radioactive labeling at one 5' end of the DNA and purification of the DNA fragment to be sequenced.
-End-label with radioactive phosphate and chemically cleave

Front 79

How do you weed out recombinant λ phage vectors?

Back 79

λ phage vectors that have failed to acquire a foreign DNA insert are unable to propagate because they are too short to form infectious phage particles.

Front 80

LacZ gene of the pUC18 plasmid cleaves what? What color is the product?

Back 80

-lacZ gene in the pUC18 plasmid (see Fig. 3-24) encodes the enzyme β-galactosidase, which cleaves the colorless compound X-gal to a blue product

Front 81

How do you weed out recombinant vectors using the LacZ gene of the pUC18 plasmid?

Back 81

-if the plasmid contains a foreign DNA insert in its polylinker region, the colonies are colorless because the insert interrupts the protein-coding sequence of the lacZ gene and no functional β-galactosidase is produced.
-If the ligation was successful, the bacterial colony will be white; if not, the colony will be blue.

Front 82

How is a cDNA library made?

Back 82

cDNA library is constructed by isolating all the cell's mRNAs and then copying them to DNA using a specialized type of DNA polymerase known as reverse transcriptase because it synthesizes DNA using RNA templates (Box 25-2). The complementary DNA (cDNA) molecules are then inserted into cloning vectors to form a cDNA library.

Front 83

Back-tracking

Back 83

The process of RNA polymerase stopping forward movement and backing up by
a few bases, ~about five bases

Front 84

Anti-termination

Back 84

A process whereby RNAP is converted to a form that is more resistant to
termination.

Front 85

β' trigger–loop

Back 85

RNAP loop that recognizes the correct rNTP substrates at the active site of RNA polymerase. It changes its conformation from a disordered loop to two α
helices when the correct rNTP enters the active site and triggers phosphodiester bond formation.

Front 86

NADFDGD

Back 86

The absolutely conserved NADFDGD motif, present in all RNA polymerases, that
is involved in coordination the active site Mg2+ ions, which are essential for
catalysis

Front 87

heptapeptide

Back 87

The YSPTSPS conserved sequence many copies of which are found on the Cterminal
domain of RNAP II. The conserved ser residues are differentially phosphorylated at various stages of transcription.

Front 88

Footprinting

Back 88

The process of determining the binding site of a protein or enzyme (RNA Polymerase) on DNA.

Front 89

Genes required for growth under oxidative stress

Back 89

Sigma^oxstress

Front 90

Genes required for growth under cold shock conditions.

Back 90

Sigma^coldshock

Front 91

Genes required for stationary phase growth

Back 91

Sigma^S

Front 92

Genes required for growth under high pressure

Back 92

Sigma^HiPressure

Front 93

ORF

Back 93

is a DNA sequence that does not contain a stop codon in a given reading frame

Front 94

Open Complex

Back 94

the complex of RNA polymerase and one DNA strand (The antisense strand)

Front 95

Closed Complex

Back 95

complex of RNA Polymerase when it is bound to the double stranded DNA

Front 96

Silencer

Back 96

Silencers are control regions of DNA that, like enhancers, may be located thousands of base pairs away from the gene they control. However, when transcription factors bind to them, expression of the gene they control is repressed.

Front 97

Activator

Back 97

DNA-binding protein that regulates one or more genes by increasing the rate of transcription. The activator may increase transcription by virtue of a connected domain which assists in the formation of the RNA polymerase holoenzyme, or may operate through a coactivator.

Front 98

Coactivator

Back 98

A coactivator is a protein that increases gene expression by binding to an activator (or transcription factor) which contains a DNA binding domain.

Front 99

Proximal elements

Back 99

the proximal sequence upstream of the gene that tends to contain primary regulatory elements

Front 100

Distal Elements

Back 100

the distal sequence upstream of the gene that may contain additional regulatory elements, often with a weaker influence than the proximal promoter

Front 101

Consensus Sequence

Back 101

a sequence of nucleotides or amino acids similar or identical between regions of homology in different but related DNA, RNA, or protein sequence.

Front 102

AAUAA

Back 102

Sequence on the 3' end that signals cleavage of mRNA by poly A polymerase some 20 nucleotides downstream of the signal.

Front 103

Downstream

Back 103

3' End

Front 104

mRNA cap

Back 104

5' End

Front 105

How do restriction enzymes protect themselves?

Back 105

EcoRI methylase adds a single methyl group to the recognition sequence.

Front 106

DNA Ligase

Back 106

carries out ligation by using ATP energy to make the phosphodiester bond between the vector and passenger.

Front 107

On the other hand, cDNA libraries (c stands for copy) are made by first converting mRNA into a DNA copy, a process known as____

Back 107

Reverse transcription