Control Of Gene Expression Flashcards

(245 cards)

1
Q

Do all cells contain the same genes?

A

Yes but different genes are expressed in each cell type

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2
Q

What is a gene from the perspective of a molecular biologist

A

A sequence of genomic DNA that encoded a single functional RNA

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3
Q

Give the 4 key steps in protein synthesis from DNA

Which steps are regulatory

A

Transcription-> splicing -> editing and export-> protein synthesis and degradation

All of them

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4
Q

Is protein synthesis from DNA compartmentalised

A

Yes

Transcription is in the nucleus and translation is in the cytoplasm

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5
Q

What are Exon’s and introns

What are promoters

A

Exon: expressed DNA segments
Introns: intervening sequences

Promoter: sequences which ensure that the gene is transcribed at the appropriate time and in the correct cell type

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6
Q

In mRNA what are coding regions flanked by

A

Untranslated regions (UTRs) at both 5’ and 3’ ends

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7
Q

Which RNAs are most abundant

A

tRNA and rRNA

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8
Q

How often do prokaryotic cells divide

A

Every 20 mins

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9
Q

How is prokaryotic DNA adapted for speed and rapid response to altered environment

A

No nucleus
No introns
mRNA is translated while still being transcribed

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10
Q

What is antitermination

A

The prokaryotic cell’s aid to fix premature termination of RNA synthesis during RNA transcription and often occurs when RNA polymerase ignores the termination signal and continues until a second signal is reached

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11
Q

What is transcription
What is it catalysed by
In what direction does it occur

A

The synthesis of single stranded RNA from a double stranded DNA template
Catalysed by RNA polymerase
Occurs in 5’ to 3’ direction

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12
Q

For any region of dsDNA what is copied by RNA in prokaryotes

A

Only one strand (the coding strand)

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13
Q

Is transcription continuous?

A

No it occurs in discrete units

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14
Q

Compare the length of the completed RNA chains to the whole bacterial DNA

What does this suggest?

A

RNA: 100 - 10,000 nucleotides
DNA: 4.7x10^6 nucleotides

RBA is copies in discrete units in the continuous DNA molecule with well defined starting and stopping points

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15
Q

Which strand on dsDNA may act as the coding strand

A

Either can

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16
Q

In bacterial, how many RNA polymerases are required

A

Only one RNA polymerase synthesises all mRNA, rRNA, and tRNA

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17
Q

How long do rRNA and tRNA molecules in bacterial cells last

A

They are very stable and persist for many generations

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18
Q

Compare the stability of rRNA, tRNA, and mRNA

A

rRNA and tRNA persist for many cell divisions due to their high stability

mRNA is rapidly degraded and replaced
The average half-life of E. coli mRNA is 2 minutes

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19
Q

What is required for prokaryotic RNA synthesis

A

DNA template to copy
riboNTPs (eg ATP, GTP)
No primer needed

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20
Q

What riboNTPs are required for prokaryotic RNA synthesis

A

APT
GTP
CTP
UTP

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21
Q

How does the precursor NTP interact with a growing RNA chain

What is the NTP usually

A

The phosphate attached to the 5’-OH terminus of the precursor NTP forms an Ester bond with the 3’-OH at the end of the growing chain with concomitant release of phosphate

ATP or GTP

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22
Q

What is on the very 5’end of an RNA chain

A

A triphosphate

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23
Q

How does the newly created RNA strand relate to the 2 strands of DNA

A

It is complementary and anti parallel to the template strand

It has the same sequence (replacing U with T) as the coding strand

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24
Q

What is the error frequency in prokaryotic RNA production

A

1 error per 10^4 nucleotides

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25
How does error frequency compare between RNA and DNA synthesis in prokaryotes
Much higher in RNA as RNA polymerase has no proof reading 3’-5’ exonuclease activity and there are no other correction mechanisms
26
What must happen to DNA before RNA can be synthesised
DNA melting
27
Discuss the structure of an RNA polymerase
Core is made of β subunit, β’ subunit, and 2 α subunits which are identical β subunits are claw-like clamps The holoenzyme also consists of a σ subunit as well
28
What is the difference between the core and holoenzyme in prokaryotic RNA Polymerase
Core binds DNA randomly The σ in the holo is limiting, ensuring the specific binding to transcription start sites (it is a promoter) This reduces non specific binding
29
Is RNA polymerase asymmetric
Yes | While there are 2 α subunits, the other subunits are all different
30
What is the first and principal stage in gene expression Is this for eukaryotes or prokaryotic cells?
Transcription Both
31
Where does transcription start
Selected sites called promoters (where the RNA polymerase binds to begin transcription)
32
What is the closed complex in transcription
When the holoenzyme first binds to the promoter with any opening of the DNA
33
How much does an RNA polymerase open DNA What is this stage called
~15bp Open complex
34
What happens after open complex is achieved by prokaryotic RNA polymerase
It selects the appropriate NTP that will base pair correctly with the DNA template strand nucleotide that is in the active site at that moment
35
What does the RNA do after incorporating the ribonucleotide and eliminating the pyrophosphate
The enzyme moves on one nucleotide and repeats the process of correct NTP selection
36
What happens when the RNA chain has grown by ~6-8 nucleotides
The σ subunit is released and can join another core enzyme to intimate synthesis of another chain
37
How is the DNA reformed as RNA polymerase ploughs along
dsDNA is reformed by displacement of the newly formed RNA by the RNA polymerase’s “rudder like” action
38
How is a promoter deduced
By comparing sequences of promoters in different E. coli genes and examining whether mutations of these sequences affect binding of RNA polymerase and the efficiency of initiation of RNA synthesis at that promoter
39
How is the consensus sequence defined
By aligning all known examples if promoter sequences to maximise their homology
40
Give 2 motifs associated with RNA synthesis
-35 region and -10 region (AKA Pribnow Box)
41
Is the sequence of a promoter symmetric What does this mean
No the sequence read 5’ to 3’ on one strand is v different to the 5’ to 3’ on the other DNA strand There is just one orientation in which the enzyme can bind to DNA and these site will lie in different locations on each strand No extra info is needed to instruct the enzyme which DNA strand to copy
42
What is the numbering convention for RNA synthesis
Number the DNA base pairs from the start point of mRNA synthesis: positive numbering in one direction and negative in the other
43
Briefly, what happens at the termination of RNA synthesis
Addition of nucleotides to the growing RNA chain stops, the RNA-DNA duplex is broken and the polymerase dissociates from the DNA
44
What are the 2 types of bacterial termination sites
ρ dependant and ρ independent
45
What happens at bacterial ρ independent sites
Core enzyme terminates due to 2 structural features: 1) A GC rich hairpin 2) the hair pin is followed by a run of ~6 Us in the RNA
46
What is important about the palindrome in the DNA sequence during RNA synthesis
The RNA copies from this DNA has self complementary regions and folds into a base paired hair pin, the stem of which is GC rich and stable This is followed by a run of unpaired U residues
47
What does the stable hair pin formed by self complementary regions of RNA do?
It is believed to pause the RNA polymerase which then dissociates from DNA due to very weak associations between the rU stretch and the dA template strand
48
What is the significance of the U rich region after the stable hair pin in RNA
Allows RNA polymerase to dissociate from DNA due to the weak IMFs between the U on RNA and A on the DNA template strand
49
How is the stable hair pin in the RNA different for rho dependant termination sites What precedes the hair pin
They usually lack the U tract (still have the hairpin) A 50-90 nucleotide region with high C content
50
What is Rho and what does it do in RNA synthesis in prokaryotes
It is an ATP dependant helicase that binds to the C rich region before the stable hairpin May unwind the RNA-DNA duplex while the polymerase is paused at the hair pin
51
What does prokaryotic regulation of transcription depend on
The efficiency of the promoter and the regulatory proteins that control access of RNA polymerase to the promoter
52
Name a major control mechanism that regulates the quantity of different mRNA species
Promoter efficiency
53
Which promoters are the most efficient
Those that match the consensus most closely
54
What is an operon
A single bacterial promoter that links many genes together and controls them all
55
Which promoter is bacterial mRNA generated from
An operon as a polycistronic transcript
56
What is a polycistronic transcript
Encodes several different proteins
57
What is the most common sigma factor in E. coli Why is it called that What do sigma factors do
σ70 After its size in kDa Recognise promoter regions
58
Describe what happens to promoters in heat shock
mRNA synthesis is shut down and heat shock genes are switched on by using σ32 which directs RNA polymerase to the heat shock promoters with their unique -10 and -35 consensus sequences
59
What do heat shock genes do
Produce proteins protect the cell from this stress
60
What do bacterial regulatory proteins do
Control the frequency of initiation of RNA synthesis in response to the concentration of metabolites They can act negatively or positively
61
What do repressors do
Blocks RNA synthesis when it is bound to DNA, usually because it’s binding site overlaps that of the RNA polymerase. When the repressor is bound, the RNA polymerase cannot vain access to the promoter
62
What do positive regulators do in RNA synthesis How do they do this Give an example
Binds to a specific DNA sequence and enhances the efficiency of RNA polymerase entry, binding and initiation of transcription Providing extra recognition contacts for the RNA polymerase CAP (catabolite activator protein)
63
What are the 2 conformations or regulatory proteins How is a particular conformation stabilised
One binds to a specific sequence on the DNA at the promoter to be controlled The other does not bind to this sequence One is stabilised by the binding of a particular metabolite
64
What happens to lactose
Hydrolysed by β- galactosidase to galactose and glucose
65
How much β galactosidase will an E. coli strand have in a solution where glucose is the sole source of C and energy What happens if it is put in a solution containing lactose What does this show
There are low levels of β gal Activity of β gal increases a thousand fold within ~20 mins Bacteria adapt v quickly to surroundings by inducing the transcription of the enzyme genes needed to metabolism the available nutrients
66
What induces the synthesis of β galactosidase mRNA
A variety of β galactosides via the work of a repressor mechanism.
67
Why is [β galactosidase] low in the absence of lactose What if lactose is present
Lac repressor binds to DNA at an operator site that prevents RNA polymerase binding If lactose or non hydrolysable analogies are present the lac repressor no longer binds to DNA, thus allowing transcription
68
# Define the following: a) Constitutive | b) inducible
a) in all cells | b) Responds to stimuli
69
What words describes something that is in all cells
Constitutive
70
How many types of RNA polymerase in eukaryotes
3: | Pol I, II, III
71
What is the range of half life of eukaryotic mRNA
Minutes to tens of hours
72
Which RNA polymerase regulates mRNA
Pol II
73
Where is RNA polymerase I found What is a product of this type of RNA polymerase
In the nucleus 45S pre-rRNA
74
Where is RNA polymerase II found What is a product of this type of RNA polymerase
Nucleoplasm mRNA etc
75
Where is RNA polymerase III found What is a product of this type of RNA polymerase
Nucleoplasm Small RNAs (eg tRNA, rRNA, etc)
76
How many subunits do eukaryotic RNA polymerases have How many will initiate transcription accurately without additional transcription factors
~12 None
77
What are DNA promoters in eukaryotes
Sequences in the vicinity of the transcription start site that are required for accurate and efficient initiation of mRNA synthesis
78
What are regulatory elements in eukaryotes
Genes that are expressed with cell type specificity or induced by hormones etc
79
What 4 types of DNA elements that transcription factors bind to should you know??
Core promoter element Upstream promoter element Enhancer sequences Response elements
80
What is the common element in a variety of elements (the promoter core element)
The AT rich TATA box
81
What is the TATA box
Centred -25 base pairs from start site which is present in most Pol II promoters
82
In vitro, RNA polymerase II synthesises RNA using NTPs and a DNA template. What else is needed for accurate transcription?
Basal transcription factors | Otherwise non-specific initiation may use wrong DNA strand
83
What is the PIC
The pre initiation complex It consists of the core enzyme, a General Transcription Factor, and the regulates assembly
84
What is TFIID
A complex of TATA binding Protein (TBP) and other TBP associated factors
85
What does TBP do
Present in most eukaryotes Saddle like Binds to TATA box on minor groove by introducing a kink into the dsDNA
86
After TBP has bound to TATA what else must happen before RNA polymerase II is recruited
TFII D, A, and B must all be present Then TFIIF binds RNA polymerase II and brings it into the PIC
87
Is energy required to open DNA and keep it open
ATP needed to melt DNA but the synthesised RNA holds the bubble open
88
Is the TATA box sufficient for efficient transcription in vitro
NOPE Transcription factors are also required
89
Are transcription factors fixed
No
90
Where do transcription factors bind | What do they do generally
To the upstream promoter elements Modulate the basal level of transcription provided by RNA polymerase II and GTFs FTII A->H
91
Give the technique for Foot Printing
Introduce P-32 label to one strand of dsDNA and divide traction mixture into 2 tubes Add binding protein to one sample Perform limited nuclease digestion with DNase I on both samples Analyse sites of resulting cleaved ssDNA (labelled bands) by autoradiography Compare 2 reactions on denaturing gel to find the protected site
92
Which regions won’t show up in Foot Printing
The regions where the protein bound and protected the DNA
93
What is ChIP
Chromatin Immunoprecipitation
94
How does ChIP work
Cross link chromatin and DNA binding proteins in live cells or using formaldehyde Shear DNA into segments Purify TF and Other proteins by digestion with proteases leaving pure DNA segment Amplify DNA by PCR Can be coupled to microarray or sequencing analyses
95
What is the principal way to control gene expression in eukaryotes
Control of transcription
96
Do nucleosomes disassemble during transcription
No But promoters are nucleosome free
97
What is the evidence that nucleosomes do not disassemble during transcription but most promoters are nucleosome free?
DNAse l digestion occurs faster in genes that are being actively transcribed and DNAse I hypersensitive sites occur in promoter regions
98
What does the acetylation of lysine residues do
Acetyl group added to a free amino group to reduce the net positive charge
99
What is important about acetylated histones
Preferentially found in active genes where chromatin is less tightly packed and are therefore related to gene expression
100
What are HATs
Histone acetylases They activate gene expression Histone deacetylases inhibit gene expression
101
Give an example of a HAT
CBP (CREB binding protein)
102
How long are enhancers | What do they comprise of
~100bp A set of closely spaced transcription factor binding sites Can activate transcription from a large distance from either direction
103
How can an enhanced effect far away gene elements
DNA bending
104
Why may promoters be activated
Triggered by a stimulus eg | Heat shock, serum, heavy metals
105
Discuss the response element of CREB
cAMP response to element binding transcription factor Binds cAMP (CREB elements) Elevated cAMP levels (by adenyl cyclase) activates PKA which phosphorylates CREB
106
What does the phosphorylation of CREB do
Activates transcription activity and enables the binding of the CBP co-activator factor
107
What is CBP
CREB binding protein (a histone acetylase)
108
Does cortisol need a trans membrane receptor?
No it can pass through the plasma membrane Signal can be read inside cell
109
Why can’t GR always affect transcription When can it regulate transcription
It is regulated by nuclear import: the chaperone HSP90 keeps it in the cytoplasm When cortisol binds
110
What happens when cortisol binds to GR-HSP90 complex
GR is released and dimerises to enter nucleus
111
Why does GR dimerise before entering the nucleus
Due to symmetry of their response elements
112
What is MyoD | What is it an example of
A TF present in myoblasts which controls expression of muscle specific genes It is an example of tissue specific gene expression
113
Give an example of tissue specific gene expression
Oct-2 Regulates expression of light and heavy immunoglobulin genes in β cell lymphocytes
114
What are Hox genes
Homeotic genes that direct development of individual body parts and encode TFs
115
What happens if Hox genes mutate Give an example
The transformation of a particular body part Mutations in “Ant” genes transform insect antennae into legs
116
What can happen if the sequence of a TF is mutated in a deleterious way When can cancer result
Altered levels of mRNAs whose synthesis it normally controls If this mRNA encode proteins important in control of cell proliferation
117
When do oncogenes promote cancer
When Over expressed
118
Give an example of an oncogene which causes cancer when over expressed
c-fos Thus forms a dimer with c-Jun forming the TF “AP1” c-fos mRNA is v unstable and levels of encoded protein are low Patients with fibrous dysplasia show high levels of c-fos expression in bone lesions but not in normal surrounding tissue
119
Discuss p53
The “guardian of the genome” Acts as a tumour suppressor in normal cells It is a transcription activator Activates expression of genes whose products inhibit cellular growth
120
What activates p53 How do we know p53 is important in tumour suppression Give an example of another rumour suppressor
Cyclin dependant kinase Many of the mutant forms found in cancer have lost their sequence specific DNA binding WT1
121
Give an example of a virus causing cancer
v-Fos c-Fos caught by virus and used by retrovirus to drive cellular proliferation
122
How does p53 guard the genome
Respond to DNA damage to induce DNA repair, cell cycle arrest or even apoptosis
123
Give an example of p53 at work in holiday goers
p53 activated by UV light Tries to repair cells But can only work to a certain extent
124
What is Rb
Retinoblastoma is a co repressor Keeps gene off to prevent inappropriate cell cycling Changes chromatin to prevent transcription by de-acetylation
125
While there is no RNA proof reading, what stops proliferation/ translation of funny looking RNA in the cytoplasm
Caps
126
What is at the 5’ end of mRNA
7mG with 5’ to 5’ linkage It is special so can be easily recognised as okay
127
Discuss 7mG
GMP (from GTP) is added to the triple phosphate at the 5’ end of RNA it is added 5’ to 5’ GMP cap is methylated also
128
Why is the 5’ to 5’ linkage in 7mG important
It is a highly unusual link to allow easy recognition
129
After the 5’ cap in eukaryotic RNA what is likely to be on the 1st and 2nd base and nucleotide
Nucleotide May carry a 2-O Methyl (-O-CH3) Base may be methylated (especially if A)
130
What is at the 3’ end of eukaryotic RNA
The poly(A) tail
131
What gives control
Being able to both create and destroy
132
What is the poly(A) tail What is the consensus sequence that ends transcription
Similar to a timer for the mRNA Terminates transcription Longer the length of tail, longer RNA life AAUAA A protein complex recognises this sequence and cleaves the RNA and recruits a PAP to add many A’s to end
133
Which RNA’s don’t have a poly A tail
Histone mRNA and some viral mRNAs
134
What is the role of the poly (A) cap
Protects mRNA from 3’ exonuclease and controls degradation rate of mRNA enhances rate of translation
135
What is a disadvantage of microarrays over sequencing
You can only identify known sequences for which your microarray contains a probe
136
What are genomic DNA libraries for
To determine gene structure including introns and exons
137
How to make a DNA copy of mRNA
Purify RNA and add primer of TTT.. to poly(A) tail which ANNEALS to the DNA Using reverse transcriptase RNase removes the RNA and a DNA polymerase creates the second strand to make double helix of DNA. Then sequence in sequencing machine.
138
What is cDNA library
Only coding DNA by sequencing mRNA
139
How to compare gene expression between tissue types
Use cDNA libraries Label one tissue type eg red and the other green Mix together and put on glass slide If mRNA Is equally expressed = yellow spot (Amount of expression depends of how bright/ faint spots are) If just red, only expressed in that tissue
140
Which genes don’t have introns
Histone genes
141
How does the presence of introns vary with organism complexity
Number of introns decrease as organism becomes less complex
142
How was splicing discovered
Sharp tried to hybridise DNA with encoded mRNA to form double helix RNA does form double helices but DNA loops out as RNA is missing bits that were cut out
143
How does splicing work
Transcription is made faithfully but then introns are removed and RNA is stuck back together
144
How does the gene size of human dystrophin compare to the mRNA
The final mRNA is 0.009 of the original gene size
145
What us the spliceosome
Eg exon 1-intron-exon 2 2’ OH nucleophilic attack on phosphate connecting 1 to intron leaving 1 floating on its own 3’ OH attacks phosphate connecting intron to 2 Intron is removed leaving the lariat which is degraded and the exons are joined
146
What is a snRNPs
Small nuclear ribonucleoprotein particles (makes up sliceosome) Made of many U (RNA -RNA recognition)
147
What happens if splicing regulation goes wrong
15% of genetic diseases are caused by mutations in splicing
148
Why do we splice
Able to make lots of different proteins from a single gene if spliced differently
149
What is lupus
An autoimmune disease where patients develop antibodies against their own nuclear proteins especially snRNPs
150
What do most introns begin with
A 5’GU dinucleotide and end with AG
151
What catalyses RNA splicing
Catalytic core of the spliceosome formed by U2 and U6 smRNAs
152
What is WT1
Tumour suppressor (Wilm’s tumour) Mutations in this can lead to cancer and kidney disease as well as Gonadal dysgenesis (Frasier syndrome)
153
What is wilm’s tumour
Childhood kidney cancer
154
What is Frasier syndrome
External genitalia have female appearance despite XY genotype due to mutations in WT1 gene
155
What does the WT1 protein do
It is a transcription factor that Recognises GC- and TC- rich promoter sequences
156
Describe the structure of the WT1 protein
2 domain protein with an N terminal proline and glutamate rich activation domain and a C terminal DNA binding protein with 4 Zn fingers
157
How many variants of WT1 do normal genes contain
4
158
What do WT1 mutations lack
The DNA binding domain or parts of activation domain
159
What is the +/- KTS ratio in normal cells and in cells of a patient with Frasier syndrome
+:- 2: 1 (normal) 1: 2 (Frasier)
160
What does + and - KTS do
-KTS binds WT1 promoter DNA sequences and acts as a TF +KTS does not bind WT1 and instead functions as an RNA processing factor
161
What does KTS stand for What does it do in Frasier’s
Lysine, threonine, serine KTS between 3rd and 4th Zn finger
162
What are the best characterised RNA editions
Hydrolytic deamination Where genomically encoded C or A is converted to U and I respectively
163
What is NF1
A common hereditary disease which predisposes sufferers to tumours of the CNS and PNS
164
How does NF1 come about
Neurofibromin is encoded by the NF1 gene and is a tumour suppressor Neurofibromin contains a GAP domain which interacts with Ras to regulate signal transduction Editing of NF1 converts CGA to a UGA termination codon. This truncates the protein at the GAP domain and breaks the protein
165
What is a GAP domain
A GTPase activating protein
166
What dictates poly (A) tail shortening
Sequences in 3’ UTR of mRNA is
167
Why is a high turnover of mRNA good
Ensures a high regulatory potential and a fast response
168
Why is the degradation of c-Fos important
A rise in c fos RNA stimulates re entry of Go cells into the cell cycle but this rise is transient as the c fos is degraded An over expression of it is oncogenic
169
What induces c fos transcription
Growth factors in serum deprived cells
170
Simply, what is the major pathway of RNA degradation in eukaryotes
Deadenylation followed by de capping and 5’-3’ exonuclease action
171
Which end does exonuclease attack from
Both
172
What happens to premature stop codons in RNA Why is this important What is this pathway referred to as
They are detected and the mRNA is degraded Without degradation the mRNA might make shorter, potentially dominant negative versions of proteins Nonsense mediates decay
173
What happens to Histone mRNA prior to targeted degradation
It gets a poly (U) tail
174
Can RNA be degraded by endonucleases
Yas
175
What do microRNAs and RNAi’s do to mRNA
Destabilise it
176
Which organelles have their own genomes
Mitochondria and chloroplasts
177
How many amino acids are there How many nucleotides
20 4
178
How many triplet codons are there Which denote termination
64 There are 3 that denote termination (UGA, UAG, UAA)
179
What does it mean to say the genetic code is degenerate
Some amino acids are denoted by more than one codon This is also referred to as redundancy
180
What are sense codons
The 61 codons specifying termination
181
Which codons are stop codons/ nonsense
UGA UAA UAG
182
What do synonym codons do Eg
Specify the same amino acid UUU and UUC
183
What is the role of tRNA
To serve as adaptors between an appropriate amino acid and its codon on mRNA
184
How many types of tRNA does each cell contain
Between 45 and 100
185
Name 5 things all tRNAs have in common
1) length of 75-90 nucleotides 2) 15-20% of unusual modified bases 3) an unpaired sequence at 3’ end of CCA, whose 3’OH or 2’OH is aminoacylated 4) clover shaped in 2D but L shaped in 3D 5) acceptor arm and anticodon loop (key active sites) are at opposite ends
186
Is peptide bond formation energetically favourable
No
187
what do all proteins start with
Methionine
188
Give the 2 step process that all activating enzymes operate by
Amino acid forms a tight complex with the enzyme using ATP The correct tRNA for that amino acid replaces the enzyme making an amino acid - tRNA complex
189
What do the 2 tRNA active sites do
First discriminates between amino acids and the second proof reads
190
How many aminoacyl tRNA synthetases are there
20 | One for each amino acid
191
How is the mRNA code translated Can each species of tRNA read more than one codon
By base paring between the trinucleotide codon on the mRNA and the trinucleotide anti codon sequence on the tRNA Yes
192
Why can a given species of tRNA read more than one codon Why does this happen
First 2 bases of codon are recognised strictly according to Watson-Crick rules but it is more lenient on the 3rd base Phosphate backbone around the wobble position in tRNA is flexible so the first base on the anticodon can adopt different positions, allowing non standard base pairing between it and the 3rd codon base
193
What happens when inosine is in the tRNA wobble position
It can Code for U C or A
194
What is the proportion of RNA:protein in ribosomes
3:1
195
Are S values additive What are they
No Sedimentation coefficient expressed in Svedberg units Depends on Mr and particle shape
196
What is the S value of ribosomes engaged in elongation
70S/80S
197
What happens to ribosomes at termination What happens next
Released from mRNA to enter a pool of free subunits These will associate in inactive complexes unless a specific anti association factor binds them
198
How do you initiate protein synthesis
The small ribosomal subunit binds mRNA first and then the large subunit joins it at the initiating AUG codon to form a whole ribosome capable of elongation
199
What is polysomes
Polyribosomes Several ribosomes simultaneously translating the same mRNA molecule while each ribosome acts independently
200
What is the maximum density of a poly some
1 ribosome per 80 nucleotides
201
What direction is mRNA translated What does this mean What direction are proteins synthesised
mRNA from the 5’ end As RNA is synthesised in 5’ to 3’ direction, it can be simultaneously translated as they are made Proteins are synthesised in N to C direction
202
What do all newly synthesised proteins in bacteria start with
Formyl-methionine The formyl group is quickly removed, and the methionine group may be removed later
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What removes formyl
Deformylase
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What is the initiator codon What can it be in bacteria
AUG GUG or UUG
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What is the correct initiation site characterised by in bacteria
The presence of a polypurine tract | (5’-..GGAGG-..3’) locates nearer the 5’ end than the AUG initiation codon
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What is 5’-..GGAGG..-3’ sequence called
The Shine Dalgarno sequence
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Which subunit recognises the poly purine tract How does this happen
The 30S ribosomal subunit Base pairing between the GGAGG (RNA) and 5’..CCUCC..3’ ay the end of the 16S rRNA in the 30S subunit
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What does polycistronic mRNA
mRNA that codes for more than one protein (Max= 15 proteins) Each cistron has its own AUG codon and Shine Delgarno sequence Each is translated separately
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How common is polycistronic mRNA in eukaryotes
Eukaryotic mRNA is always monocistronic
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How do eukaryotes select the right translation initiation site
The 40S ribosomal subunit bind to the extreme 5’ end of the mRNA and there is scamming towards the 3’ end When the first AUG codon is reached, the Met tRNA already on the 40S subunit locks on the AUG codon and the 60S subunit then join
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Give 4 features of the scanning ribosome model
No CCUCC in 18S rRNA 5’ cap binds cap binding protein which recruits 40S subunit Scanning is coupled with ATP hydrolysis Different start sites are used to generate different proteins from one mRNA
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Eukaryotic translation is cap - independent True or false
False It requires the 5’ m7G cap on the RNA
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What is advantageous about eukaryotic translation being cap dependant
Initiation factors that bind to the cap structure also bind the small ribosomal subunit and thus direct its recruitment to the 5’ end of mRNA
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What was internal initiation of translation first discovered in How does it work
Picornaviruses (small RNA viruses) eg polio Their RNA is uncapped and their 5’ untranslated regions are v long with multiple AUG codons Translated by internal initiation whereby an IRES in the 5’UTR directs binding of the ribosome to the mRNA to the correct initiator AUG
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What is an IRES and how is it experimentally defined
Internal ribosomal entry site By dicistronic assays
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How does a dicistronic assay work
A potential sequence is inserted between protein coding frames of 2 reporter genes in a plasmid vector Normal eukaryotes would not translate the second reporter gene Second gene translation occurs if the inserted sequence can direct ribosome entry directly
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Are IRES heterogenous
Yes
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Give an example of use of IRES in medicine
Hepatitis C IRES is a major drug target
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What is the advantage of having an IRES
Viruses (eg Polio) is encode a protease which cleaves one of the cap binding factors in 2 pieces, compromising host cell protein synthesis. This allows viruses to hijack cellular translation machinery
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Do eukaryotes have IRESes?
Yes They direct translation in situations where cap dependant translation is reduced Eg during mitosis, apoptosis and under stress
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What is charged tRNA
tRNA with an amino acyl ester
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What signifies that the ribosome is ready for the elongation phase of protein synthesis
Formation of the 80S or 70S initiation complex
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What are nascent polypeptide chains found as
Peptidyl tRNA where the C terminus of the protein is bonded to the tRNA (which is non covalently bound to the ribosome)
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What are the 2 ribosomal sites for binding charged tRNA
``` P site (peptidyl tRNA) A site (amino acyl tRNA) ```
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Where is the anticodon of Met-tRNA locates What does it pair with What does this define
P site The initiating AUG codon The reading frame and sets the stage for elongation
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Where do most amino acyl tRNAs bond What is required
A site Elongation Factor protein and GTP After GTP hydrolysis the EFTu-GDP form of elongation factor dissociates from the ribosome
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What are the elongation factors in bacteria and eukaryotes
B : EFTu E: eEF-1
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What catalyses peptide bond formation What is required
Peptidyl transferase activity of the large ribosomal subunit No energy needed Must be formed by transfer of a peptide from the P site to an amino group on the A site MOT VICE VERSA
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What is translocation
When ribosomes move along the mRNA by 3 nucleotides and peptide bond synthesis
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What does translocation require
Requires the action of another elongation factor (EFG in bacteria and eEF-2 in eukaryotes) and GTP
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What does translocation do
Not only moves the ribosome but also shifts the peptidyl tRNA from A site to P site whilst ejecting the deaceylated tRNA from the P site
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How does discharged tRNA leave the ribosome
There is an additional 3rd tRNA binding site the E site (E for exit) near the P site
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True or false Gene amplification involves acquisition of mutations in the encoded protein
False
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Does PCR require RNA primers
No
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Does HSP90 (heat shock protein 90 kDa) control gene expression
Not directly
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Which experimental approach is most suitable to identify all of the binding sites for a specific DNA binding protein in the genome
ChIP
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What is a distinguishing feature of the lariat
2’ to 5’ phosphodiester bond
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What is the U1 snRNA binding site
5’ splice site
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Which is rho dependent and which is rho independent
Independent: GC hairpin with 6+ U after Dependant: GC hairpin preceded by many C - rho binds to C and unwinds DNA using ATP
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How is CREB involved in gene expression
Raised cAMP (eg from adrenaline) increases PKA PKA phosphorylates CREB, allowing CBP to bind Histone can be actylated
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Name a specific protein transcription regulation
Ferritin mRNA and IRP ferritin prevents Fe build up Ferritin mRNA contains a 5’ hairpin (IRE/ iron response element) When IRE is bound by IRP (iron regulatory protein), 40S ribosome cannot bind to cap so translation is prevented, allowing Fe accumulation When Fe is high, IRP has low affinity to IRE, so ferritin synthesis can proceed
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What are the 3 steps of ubiquitination
E1 - uses ATP to link C terminal Gly in Ub to a SH group in E1, forming a thioester bond E2- Ub conjugating enzyme - takes Ub from E1 and ligates to its own SH group E3 - Ub ligase - takes Ub from E2 and ligates it to the e-NH2 groups of lysines in the protein destined for destruction
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How are more Ub added to an Ub
By linking C terminal glycine of the first Ub to internal lysine of the next
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Name 3 destruction sequences
N - end rule: 5αα added to N terminal PEST - motifs containing Proline (P), glutamate (E), Serine (S) and threonine (T) are phosphorylated on S or T for destruction D box - N terminus of cyclins - required for Cyclin ubiquitination
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Where are IRES sequences found
5’UTR region of RNA viruses | Also found in mRNA of eukaryotes (for stress survival )