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MIMS > Control Of Gene Expression > Flashcards

Control Of Gene Expression Flashcards

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
Q

How does error frequency compare between RNA and DNA synthesis in prokaryotes

A

Much higher in RNA as RNA polymerase has no proof reading 3’-5’ exonuclease activity and there are no other correction mechanisms

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26
Q

What must happen to DNA before RNA can be synthesised

A

DNA melting

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27
Q

Discuss the structure of an RNA polymerase

A

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

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28
Q

What is the difference between the core and holoenzyme in prokaryotic RNA Polymerase

A

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

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29
Q

Is RNA polymerase asymmetric

A

Yes

While there are 2 α subunits, the other subunits are all different

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30
Q

What is the first and principal stage in gene expression

Is this for eukaryotes or prokaryotic cells?

A

Transcription

Both

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31
Q

Where does transcription start

A

Selected sites called promoters (where the RNA polymerase binds to begin transcription)

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32
Q

What is the closed complex in transcription

A

When the holoenzyme first binds to the promoter with any opening of the DNA

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33
Q

How much does an RNA polymerase open DNA

What is this stage called

A

~15bp

Open complex

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34
Q

What happens after open complex is achieved by prokaryotic RNA polymerase

A

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

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35
Q

What does the RNA do after incorporating the ribonucleotide and eliminating the pyrophosphate

A

The enzyme moves on one nucleotide and repeats the process of correct NTP selection

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36
Q

What happens when the RNA chain has grown by ~6-8 nucleotides

A

The σ subunit is released and can join another core enzyme to intimate synthesis of another chain

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37
Q

How is the DNA reformed as RNA polymerase ploughs along

A

dsDNA is reformed by displacement of the newly formed RNA by the RNA polymerase’s “rudder like” action

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38
Q

How is a promoter deduced

A

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

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39
Q

How is the consensus sequence defined

A

By aligning all known examples if promoter sequences to maximise their homology

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40
Q

Give 2 motifs associated with RNA synthesis

A

-35 region and -10 region (AKA Pribnow Box)

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41
Q

Is the sequence of a promoter symmetric

What does this mean

A

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

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42
Q

What is the numbering convention for RNA synthesis

A

Number the DNA base pairs from the start point of mRNA synthesis: positive numbering in one direction and negative in the other

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43
Q

Briefly, what happens at the termination of RNA synthesis

A

Addition of nucleotides to the growing RNA chain stops, the RNA-DNA duplex is broken and the polymerase dissociates from the DNA

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44
Q

What are the 2 types of bacterial termination sites

A

ρ dependant and ρ independent

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45
Q

What happens at bacterial ρ independent sites

A

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

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46
Q

What is important about the palindrome in the DNA sequence during RNA synthesis

A

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

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47
Q

What does the stable hair pin formed by self complementary regions of RNA do?

A

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

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48
Q

What is the significance of the U rich region after the stable hair pin in RNA

A

Allows RNA polymerase to dissociate from DNA due to the weak IMFs between the U on RNA and A on the DNA template strand

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49
Q

How is the stable hair pin in the RNA different for rho dependant termination sites

What precedes the hair pin

A

They usually lack the U tract (still have the hairpin)

A 50-90 nucleotide region with high C content

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50
Q

What is Rho and what does it do in RNA synthesis in prokaryotes

A

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

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51
Q

What does prokaryotic regulation of transcription depend on

A

The efficiency of the promoter and the regulatory proteins that control access of RNA polymerase to the promoter

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52
Q

Name a major control mechanism that regulates the quantity of different mRNA species

A

Promoter efficiency

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53
Q

Which promoters are the most efficient

A

Those that match the consensus most closely

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54
Q

What is an operon

A

A single bacterial promoter that links many genes together and controls them all

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55
Q

Which promoter is bacterial mRNA generated from

A

An operon as a polycistronic transcript

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56
Q

What is a polycistronic transcript

A

Encodes several different proteins

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57
Q

What is the most common sigma factor in E. coli

Why is it called that

What do sigma factors do

A

σ70

After its size in kDa

Recognise promoter regions

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58
Q

Describe what happens to promoters in heat shock

A

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

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59
Q

What do heat shock genes do

A

Produce proteins protect the cell from this stress

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60
Q

What do bacterial regulatory proteins do

A

Control the frequency of initiation of RNA synthesis in response to the concentration of metabolites

They can act negatively or positively

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61
Q

What do repressors do

A

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

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62
Q

What do positive regulators do in RNA synthesis

How do they do this

Give an example

A

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)

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63
Q

What are the 2 conformations or regulatory proteins

How is a particular conformation stabilised

A

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

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64
Q

What happens to lactose

A

Hydrolysed by β- galactosidase to galactose and glucose

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65
Q

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

A

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

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66
Q

What induces the synthesis of β galactosidase mRNA

A

A variety of β galactosides via the work of a repressor mechanism.

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67
Q

Why is [β galactosidase] low in the absence of lactose

What if lactose is present

A

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

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68
Q

Define the following:

a) Constitutive

b) inducible

A

a) in all cells

b) Responds to stimuli

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69
Q

What words describes something that is in all cells

A

Constitutive

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70
Q

How many types of RNA polymerase in eukaryotes

A

3:

Pol I, II, III

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71
Q

What is the range of half life of eukaryotic mRNA

A

Minutes to tens of hours

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72
Q

Which RNA polymerase regulates mRNA

A

Pol II

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73
Q

Where is RNA polymerase I found

What is a product of this type of RNA polymerase

A

In the nucleus

45S pre-rRNA

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74
Q

Where is RNA polymerase II found

What is a product of this type of RNA polymerase

A

Nucleoplasm

mRNA etc

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75
Q

Where is RNA polymerase III found

What is a product of this type of RNA polymerase

A

Nucleoplasm

Small RNAs (eg tRNA, rRNA, etc)

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76
Q

How many subunits do eukaryotic RNA polymerases have

How many will initiate transcription accurately without additional transcription factors

A

~12

None

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77
Q

What are DNA promoters in eukaryotes

A

Sequences in the vicinity of the transcription start site that are required for accurate and efficient initiation of mRNA synthesis

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78
Q

What are regulatory elements in eukaryotes

A

Genes that are expressed with cell type specificity or induced by hormones etc

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79
Q

What 4 types of DNA elements that transcription factors bind to should you know??

A

Core promoter element
Upstream promoter element
Enhancer sequences
Response elements

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80
Q

What is the common element in a variety of elements (the promoter core element)

A

The AT rich TATA box

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81
Q

What is the TATA box

A

Centred -25 base pairs from start site which is present in most Pol II promoters

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82
Q

In vitro, RNA polymerase II synthesises RNA using NTPs and a DNA template. What else is needed for accurate transcription?

A

Basal transcription factors

Otherwise non-specific initiation may use wrong DNA strand

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83
Q

What is the PIC

A

The pre initiation complex

It consists of the core enzyme, a General Transcription Factor, and the regulates assembly

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84
Q

What is TFIID

A

A complex of TATA binding Protein (TBP) and other TBP associated factors

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85
Q

What does TBP do

A

Present in most eukaryotes
Saddle like
Binds to TATA box on minor groove by introducing a kink into the dsDNA

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86
Q

After TBP has bound to TATA what else must happen before RNA polymerase II is recruited

A

TFII D, A, and B must all be present

Then TFIIF binds RNA polymerase II and brings it into the PIC

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87
Q

Is energy required to open DNA and keep it open

A

ATP needed to melt DNA but the synthesised RNA holds the bubble open

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88
Q

Is the TATA box sufficient for efficient transcription in vitro

A

NOPE

Transcription factors are also required

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89
Q

Are transcription factors fixed

A

No

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90
Q

Where do transcription factors bind

What do they do generally

A

To the upstream promoter elements

Modulate the basal level of transcription provided by RNA polymerase II and GTFs FTII A->H

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91
Q

Give the technique for Foot Printing

A

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

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92
Q

Which regions won’t show up in Foot Printing

A

The regions where the protein bound and protected the DNA

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93
Q

What is ChIP

A

Chromatin Immunoprecipitation

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94
Q

How does ChIP work

A

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

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95
Q

What is the principal way to control gene expression in eukaryotes

A

Control of transcription

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96
Q

Do nucleosomes disassemble during transcription

A

No

But promoters are nucleosome free

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97
Q

What is the evidence that nucleosomes do not disassemble during transcription but most promoters are nucleosome free?

A

DNAse l digestion occurs faster in genes that are being actively transcribed and DNAse I hypersensitive sites occur in promoter regions

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98
Q

What does the acetylation of lysine residues do

A

Acetyl group added to a free amino group to reduce the net positive charge

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99
Q

What is important about acetylated histones

A

Preferentially found in active genes where chromatin is less tightly packed and are therefore related to gene expression

100
Q

What are HATs

A

Histone acetylases

They activate gene expression

Histone deacetylases inhibit gene expression

101
Q

Give an example of a HAT

A

CBP (CREB binding protein)

102
Q

How long are enhancers

What do they comprise of

A

~100bp

A set of closely spaced transcription factor binding sites

Can activate transcription from a large distance from either direction

103
Q

How can an enhanced effect far away gene elements

A

DNA bending

104
Q

Why may promoters be activated

A

Triggered by a stimulus eg

Heat shock, serum, heavy metals

105
Q

Discuss the response element of CREB

A

cAMP response to element binding transcription factor
Binds cAMP (CREB elements)
Elevated cAMP levels (by adenyl cyclase) activates PKA which phosphorylates CREB

106
Q

What does the phosphorylation of CREB do

A

Activates transcription activity and enables the binding of the CBP co-activator factor

107
Q

What is CBP

A

CREB binding protein (a histone acetylase)

108
Q

Does cortisol need a trans membrane receptor?

A

No it can pass through the plasma membrane

Signal can be read inside cell

109
Q

Why can’t GR always affect transcription

When can it regulate transcription

A

It is regulated by nuclear import: the chaperone HSP90 keeps it in the cytoplasm

When cortisol binds

110
Q

What happens when cortisol binds to GR-HSP90 complex

A

GR is released and dimerises to enter nucleus

111
Q

Why does GR dimerise before entering the nucleus

A

Due to symmetry of their response elements

112
Q

What is MyoD

What is it an example of

A

A TF present in myoblasts which controls expression of muscle specific genes

It is an example of tissue specific gene expression

113
Q

Give an example of tissue specific gene expression

A

Oct-2

Regulates expression of light and heavy immunoglobulin genes in β cell lymphocytes

114
Q

What are Hox genes

A

Homeotic genes that direct development of individual body parts and encode TFs

115
Q

What happens if Hox genes mutate

Give an example

A

The transformation of a particular body part

Mutations in “Ant” genes transform insect antennae into legs

116
Q

What can happen if the sequence of a TF is mutated in a deleterious way

When can cancer result

A

Altered levels of mRNAs whose synthesis it normally controls

If this mRNA encode proteins important in control of cell proliferation

117
Q

When do oncogenes promote cancer

A

When Over expressed

118
Q

Give an example of an oncogene which causes cancer when over expressed

A

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
Q

Discuss p53

A

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
Q

What activates p53

How do we know p53 is important in tumour suppression

Give an example of another rumour suppressor

A

Cyclin dependant kinase

Many of the mutant forms found in cancer have lost their sequence specific DNA binding

WT1

121
Q

Give an example of a virus causing cancer

A

v-Fos

c-Fos caught by virus and used by retrovirus to drive cellular proliferation

122
Q

How does p53 guard the genome

A

Respond to DNA damage to induce DNA repair, cell cycle arrest or even apoptosis

123
Q

Give an example of p53 at work in holiday goers

A

p53 activated by UV light
Tries to repair cells
But can only work to a certain extent

124
Q

What is Rb

A

Retinoblastoma is a co repressor
Keeps gene off to prevent inappropriate cell cycling

Changes chromatin to prevent transcription by de-acetylation

125
Q

While there is no RNA proof reading, what stops proliferation/ translation of funny looking RNA in the cytoplasm

A

Caps

126
Q

What is at the 5’ end of mRNA

A

7mG with 5’ to 5’ linkage

It is special so can be easily recognised as okay

127
Q

Discuss 7mG

A

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
Q

Why is the 5’ to 5’ linkage in 7mG important

A

It is a highly unusual link to allow easy recognition

129
Q

After the 5’ cap in eukaryotic RNA what is likely to be on the 1st and 2nd base and nucleotide

A

Nucleotide May carry a 2-O Methyl (-O-CH3)

Base may be methylated (especially if A)

130
Q

What is at the 3’ end of eukaryotic RNA

A

The poly(A) tail

131
Q

What gives control

A

Being able to both create and destroy

132
Q

What is the poly(A) tail

What is the consensus sequence that ends transcription

A

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
Q

Which RNA’s don’t have a poly A tail

A

Histone mRNA and some viral mRNAs

134
Q

What is the role of the poly (A) cap

A

Protects mRNA from 3’ exonuclease and controls degradation rate of mRNA

enhances rate of translation

135
Q

What is a disadvantage of microarrays over sequencing

A

You can only identify known sequences for which your microarray contains a probe

136
Q

What are genomic DNA libraries for

A

To determine gene structure including introns and exons

137
Q

How to make a DNA copy of mRNA

A

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
Q

What is cDNA library

A

Only coding DNA by sequencing mRNA

139
Q

How to compare gene expression between tissue types

A

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
Q

Which genes don’t have introns

A

Histone genes

141
Q

How does the presence of introns vary with organism complexity

A

Number of introns decrease as organism becomes less complex

142
Q

How was splicing discovered

A

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
Q

How does splicing work

A

Transcription is made faithfully but then introns are removed and RNA is stuck back together

144
Q

How does the gene size of human dystrophin compare to the mRNA

A

The final mRNA is 0.009 of the original gene size

145
Q

What us the spliceosome

A

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
Q

What is a snRNPs

A

Small nuclear ribonucleoprotein particles (makes up sliceosome)

Made of many U (RNA -RNA recognition)

147
Q

What happens if splicing regulation goes wrong

A

15% of genetic diseases are caused by mutations in splicing

148
Q

Why do we splice

A

Able to make lots of different proteins from a single gene if spliced differently

149
Q

What is lupus

A

An autoimmune disease where patients develop antibodies against their own nuclear proteins especially snRNPs

150
Q

What do most introns begin with

A

A 5’GU dinucleotide and end with AG

151
Q

What catalyses RNA splicing

A

Catalytic core of the spliceosome formed by U2 and U6 smRNAs

152
Q

What is WT1

A

Tumour suppressor (Wilm’s tumour)

Mutations in this can lead to cancer and kidney disease as well as Gonadal dysgenesis (Frasier syndrome)

153
Q

What is wilm’s tumour

A

Childhood kidney cancer

154
Q

What is Frasier syndrome

A

External genitalia have female appearance despite XY genotype due to mutations in WT1 gene

155
Q

What does the WT1 protein do

A

It is a transcription factor that Recognises GC- and TC- rich promoter sequences

156
Q

Describe the structure of the WT1 protein

A

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
Q

How many variants of WT1 do normal genes contain

A

4

158
Q

What do WT1 mutations lack

A

The DNA binding domain or parts of activation domain

159
Q

What is the +/- KTS ratio in normal cells and in cells of a patient with Frasier syndrome

A

+:-

2: 1 (normal)
1: 2 (Frasier)

160
Q

What does + and - KTS do

A

-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
Q

What does KTS stand for

What does it do in Frasier’s

A

Lysine, threonine, serine

KTS between 3rd and 4th Zn finger

162
Q

What are the best characterised RNA editions

A

Hydrolytic deamination

Where genomically encoded C or A is converted to U and I respectively

163
Q

What is NF1

A

A common hereditary disease which predisposes sufferers to tumours of the CNS and PNS

164
Q

How does NF1 come about

A

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
Q

What is a GAP domain

A

A GTPase activating protein

166
Q

What dictates poly (A) tail shortening

A

Sequences in 3’ UTR of mRNA is

167
Q

Why is a high turnover of mRNA good

A

Ensures a high regulatory potential and a fast response

168
Q

Why is the degradation of c-Fos important

A

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
Q

What induces c fos transcription

A

Growth factors in serum deprived cells

170
Q

Simply, what is the major pathway of RNA degradation in eukaryotes

A

Deadenylation followed by de capping and 5’-3’ exonuclease action

171
Q

Which end does exonuclease attack from

A

Both

172
Q

What happens to premature stop codons in RNA

Why is this important

What is this pathway referred to as

A

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
Q

What happens to Histone mRNA prior to targeted degradation

A

It gets a poly (U) tail

174
Q

Can RNA be degraded by endonucleases

A

Yas

175
Q

What do microRNAs and RNAi’s do to mRNA

A

Destabilise it

176
Q

Which organelles have their own genomes

A

Mitochondria and chloroplasts

177
Q

How many amino acids are there

How many nucleotides

A

20

4

178
Q

How many triplet codons are there

Which denote termination

A

64

There are 3 that denote termination (UGA, UAG, UAA)

179
Q

What does it mean to say the genetic code is degenerate

A

Some amino acids are denoted by more than one codon

This is also referred to as redundancy

180
Q

What are sense codons

A

The 61 codons specifying termination

181
Q

Which codons are stop codons/ nonsense

A

UGA
UAA
UAG

182
Q

What do synonym codons do

Eg

A

Specify the same amino acid

UUU and UUC

183
Q

What is the role of tRNA

A

To serve as adaptors between an appropriate amino acid and its codon on mRNA

184
Q

How many types of tRNA does each cell contain

A

Between 45 and 100

185
Q

Name 5 things all tRNAs have in common

A

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
Q

Is peptide bond formation energetically favourable

A

No

187
Q

what do all proteins start with

A

Methionine

188
Q

Give the 2 step process that all activating enzymes operate by

A

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
Q

What do the 2 tRNA active sites do

A

First discriminates between amino acids and the second proof reads

190
Q

How many aminoacyl tRNA synthetases are there

A

20

One for each amino acid

191
Q

How is the mRNA code translated

Can each species of tRNA read more than one codon

A

By base paring between the trinucleotide codon on the mRNA and the trinucleotide anti codon sequence on the tRNA

Yes

192
Q

Why can a given species of tRNA read more than one codon

Why does this happen

A

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
Q

What happens when inosine is in the tRNA wobble position

A

It can Code for U C or A

194
Q

What is the proportion of RNA:protein in ribosomes

A

3:1

195
Q

Are S values additive

What are they

A

No

Sedimentation coefficient expressed in Svedberg units
Depends on Mr and particle shape

196
Q

What is the S value of ribosomes engaged in elongation

A

70S/80S

197
Q

What happens to ribosomes at termination

What happens next

A

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
Q

How do you initiate protein synthesis

A

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
Q

What is polysomes

A

Polyribosomes

Several ribosomes simultaneously translating the same mRNA molecule while each ribosome acts independently

200
Q

What is the maximum density of a poly some

A

1 ribosome per 80 nucleotides

201
Q

What direction is mRNA translated
What does this mean

What direction are proteins synthesised

A

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
Q

What do all newly synthesised proteins in bacteria start with

A

Formyl-methionine

The formyl group is quickly removed, and the methionine group may be removed later

203
Q

What removes formyl

A

Deformylase

204
Q

What is the initiator codon

What can it be in bacteria

A

AUG

GUG or UUG

205
Q

What is the correct initiation site characterised by in bacteria

A

The presence of a polypurine tract

(5’-..GGAGG-..3’) locates nearer the 5’ end than the AUG initiation codon

206
Q

What is 5’-..GGAGG..-3’ sequence called

A

The Shine Dalgarno sequence

207
Q

Which subunit recognises the poly purine tract

How does this happen

A

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

208
Q

What does polycistronic mRNA

A

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

209
Q

How common is polycistronic mRNA in eukaryotes

A

Eukaryotic mRNA is always monocistronic

210
Q

How do eukaryotes select the right translation initiation site

A

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

211
Q

Give 4 features of the scanning ribosome model

A

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

212
Q

Eukaryotic translation is cap - independent

True or false

A

False

It requires the 5’ m7G cap on the RNA

213
Q

What is advantageous about eukaryotic translation being cap dependant

A

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

214
Q

What was internal initiation of translation first discovered in

How does it work

A

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

215
Q

What is an IRES and how is it experimentally defined

A

Internal ribosomal entry site

By dicistronic assays

216
Q

How does a dicistronic assay work

A

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

217
Q

Are IRES heterogenous

A

Yes

218
Q

Give an example of use of IRES in medicine

A

Hepatitis C IRES is a major drug target

219
Q

What is the advantage of having an IRES

A

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

220
Q

Do eukaryotes have IRESes?

A

Yes
They direct translation in situations where cap dependant translation is reduced
Eg during mitosis, apoptosis and under stress

221
Q

What is charged tRNA

A

tRNA with an amino acyl ester

222
Q

What signifies that the ribosome is ready for the elongation phase of protein synthesis

A

Formation of the 80S or 70S initiation complex

223
Q

What are nascent polypeptide chains found as

A

Peptidyl tRNA where the C terminus of the protein is bonded to the tRNA (which is non covalently bound to the ribosome)

224
Q

What are the 2 ribosomal sites for binding charged tRNA

A 
P site (peptidyl tRNA)
A site (amino acyl tRNA)
225
Q

Where is the anticodon of Met-tRNA locates

What does it pair with

What does this define

A

P site
The initiating AUG codon

The reading frame and sets the stage for elongation

226
Q

Where do most amino acyl tRNAs bond

What is required

A

A site

Elongation Factor protein and GTP

After GTP hydrolysis the EFTu-GDP form of elongation factor dissociates from the ribosome

227
Q

What are the elongation factors in bacteria and eukaryotes

A

B : EFTu

E: eEF-1

228
Q

What catalyses peptide bond formation

What is required

A

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

229
Q

What is translocation

A

When ribosomes move along the mRNA by 3 nucleotides and peptide bond synthesis

230
Q

What does translocation require

A

Requires the action of another elongation factor (EFG in bacteria and eEF-2 in eukaryotes) and GTP

231
Q

What does translocation do

A

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

232
Q

How does discharged tRNA leave the ribosome

A

There is an additional 3rd tRNA binding site the E site (E for exit) near the P site

233
Q

True or false

Gene amplification involves acquisition of mutations in the encoded protein

A

False

234
Q

Does PCR require RNA primers

A

No

235
Q

Does HSP90 (heat shock protein 90 kDa) control gene expression

A

Not directly

236
Q

Which experimental approach is most suitable to identify all of the binding sites for a specific DNA binding protein in the genome

A

ChIP

237
Q

What is a distinguishing feature of the lariat

A

2’ to 5’ phosphodiester bond

238
Q

What is the U1 snRNA binding site

A

5’ splice site

239
Q

Which is rho dependent and which is rho independent

A

Independent: GC hairpin with 6+ U after

Dependant: GC hairpin preceded by many C - rho binds to C and unwinds DNA using ATP

240
Q

How is CREB involved in gene expression

A

Raised cAMP (eg from adrenaline) increases PKA
PKA phosphorylates CREB, allowing CBP to bind
Histone can be actylated

241
Q

Name a specific protein transcription regulation

A

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

242
Q

What are the 3 steps of ubiquitination

A

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

243
Q

How are more Ub added to an Ub

A

By linking C terminal glycine of the first Ub to internal lysine of the next

244
Q

Name 3 destruction sequences

A

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

245
Q

Where are IRES sequences found

A

5’UTR region of RNA viruses

Also found in mRNA of eukaryotes (for stress survival )

MIMS (12 decks)

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