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Biomed > Gene expression and its control BI501 > Flashcards

Gene expression and its control BI501 Flashcards

1
Q

Name the 3 characteristics of mRNA that make it different to DNA

A

1) single stranded
2) ribose sugar instead of deoxyribose
3) Uracil replaces Thymine

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2
Q

That is the name of the region that regulates transcription?

A

Promoter region

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3
Q

What are the 4 stages of transcription?

A

1) Template recognition
2) Initiation of transcription (Steps 1&2 are INITIATION)
3) ELONGATION
4) TERMINATION

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4
Q

Name 3 characteristics of prokaryotes

A

Single celled
lack membrane bound organelles
divided into bacteria and archea

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5
Q

Name 3 differences between gene expression if prokaryotes and eukaryotes

A

Pro: transcription and translation occur in same place
Eu: Transcription in nucleus, translation in cytoplasm

Pro: single type of RNAP used to produce mRNA, rRNA and tRNA
Eu: 3 RNAP’s

Pro: Genes organised into operons
Eu: No operons

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6
Q

What are operons and how are they regulated?

A

Operons are clusters of co-regulated genes that are structurally or metabolically similar
Regulated so the gene clusters are either all on or all off

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7
Q

How are proteins produced using operons?

A

Genes are transcribed into a single RNA (polycistronic) that is translated into different proteins

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8
Q

What 2 things does transcription require?

A

1) cis-acting elements (promoter)

2) trans-acting elements (RNAP and transcription factors)

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9
Q

Where are bacterial promoters typically located?

A

Upstream or at the 5’ end of the transcription initiation site

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10
Q

The promoter sequence defines both the direction of transcription and which strand will be transcribed. What is this strand known as?

A

Sense strand

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11
Q

What are the 5 motifs in prokaryotic promoters and what part of the RNAP recognises each?

A

> the -35
the extended -10
the -10
discriminator motifs (all recognised by sigma factor)
UP element (recognised by the C-terminal domain of the RNAalpha-subunit)

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12
Q

What does the RNAP holoenzyme consist of?

A

Core enzyme and the sigma factor

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13
Q

What is the core enzyme made up of?

A
  • 2 alpha subunits
  • 2 beta subunits
  • 1 omega subunit
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14
Q

What are the functions of the beta subunits?

A

Make up the “catalytic center”

  • Primary channel which DNA passes through
  • Secondary channel which substrate ribonucleotides pass through
  • Exit channel that the RNA leaves through
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15
Q

What are the functions of the dimer formed by 2 alpha subunits?

A
  • serves as scaffold for assembly of core enzyme
  • contribute to promoter recognition
  • bind some activators
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16
Q

What is the function of the omega subunit?

A

Plays a role in enzyme assembly

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17
Q

What are the 2 most important functions of the sigma factors?

A

1) Directing the catalytic core of the RNAP to the appropriate transcription start sites
2) Suppressing Nonspecific Transcription Initiation

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18
Q

Which sigma factor domains have DNA binding elements and which prokaryotic binding motif do they bind to?

A
  • sigma4 binds the -35 motif
  • sigma3 binds the extended -10 motif
  • sigma2 binds to the -10 and discriminator motifs
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19
Q

Can free sigma factors bind to DNA promoters?

A

No

Sigma factors must be coupled with the core enzyme in order to interact with the promoter

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20
Q

What are the 2 mechanisms that contribute to suppressing the DNA-binding capacity of free sigma factors?

A

1) Conformational restriction

2) Auto-inhibitory restriction

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21
Q

In what 2 ways do sigma factors suppress nonspecific transcription initiation?

A

1) Free sigma factors suppress the core enzyme do it doesn’t initiate transcription at non promoter DNA sites
2) N-terminal sigma1.1 domain suppresses nonspecific DNA binding in the RNAP active site cleft

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22
Q

What is the transcription bubble?

A

RNAP unwinds 13 bp of DNA after it locates a promoter through recognition of DNA motifs

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23
Q

What occurs after a transcription bubble is formed?

A

Nucleotides are incorporated into RNA and a ternary complex is formed (complex containing RNA, DNA and the enzyme)

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24
Q

How are oligonucleotides produced and typically how long are they?

A

Produced by repeated cycles of abortive initiation

approx 20nt long

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25
Q

At what point is the sigma factor released from the RNAP?

A

When the RNA chain reaches approx 10 bases long

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26
Q

What are the 2 types of termination?

A

1) Intrinsic termination

2) Rho-dependent termination

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27
Q

Describe intrinsic termination

A

For it to occur, the RNA must contain a G+C-rich region that can fold into a hairpin
G+C-rich region must be followed by 7 U’s located between the hairpin an the termination site

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28
Q

Describe Rho-dependant termination

A
  • Rho is a protein that causes transcription termination
  • Rho binds to rut site upstream of site of termination
  • Rho tracks along RNA until it meets RNAP
  • When RNAP reaches termination site, rho freezes the structure of RNAP and destabilises it causing it to release the RNA
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29
Q

Name the 3 stages in which gene expression can be controlled?

A

1) Transcription
2) RNA processing
3) Translation

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30
Q

What effect will both an activator and a suppressor have when bound to the operator?

A

Activator - Initiate transcription

Repressor - Repress transcription

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31
Q

What is positive regulation?

A

When the binding of an activator is a necessary prerequisite for transcription to initiate

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32
Q

What is negative regulation?

A

When the prevention binding of an activator is a necessary prerequisite for transcription to initiate

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33
Q

How does an activator initiate transcription (1 way) and how does a repressor repress transcription (2 ways)

A

Activator - helps tether RNAP to promoter
Repressor - physically interfering with RNAP binding
- Impeding elongation

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34
Q

What 2 states can activators and repressors exist in?

A
  • DNA binding

- Non DNA binding

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35
Q

How do activators and repressors achieve 2 states?

A

Binding of allosteric effectors (inducers and co-repressors) to allosteric sites on activator/repressor

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36
Q

How is transcription initiation regulated in prokaryotes?

A

Competition of sigma factors - different sigma factors activated by different mechanisms

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37
Q

What is the lack operon?

A

Cluster of genes required for metabolism of lactose

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38
Q

When is the lac operon on and off?

A

ON in presence of lactose

OFF in presence of glucose

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39
Q

What are the 3 genes in the lac operon and what does each do?

A

lacZ - codes for beta-galactosidase

    - B-G cleaves lactose to glucose and galactose
    - Also produces 1-6-allolactose, important in        regulation

lacY - codes for protein allowing lactose to be transported into cell

lacA - codes for enzyme that transfers an acetyl group from acetyl-coA to lactose

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40
Q

What gene codes for the lac repressor?

A

lac l gene

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41
Q

What does the lac repressor do?

A

Represses lacZYA transcription

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42
Q

What are the 2 recognition sites on the lac repressor and what do they bind to?

A
  • One that binds to DNA

- Allosteric site that binds the lactose allosteric effector (allolactose)

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43
Q

What occurs when allolactose is present?

A

Binds to allosteric site on lac repressor, causes conformational change that inactivates DNA binding activity of repressor
lacZYA transcription can occur

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44
Q

What is the structure of the lac l protein?

A
  • DNA binding domain contains a helix-turn-helix motif
  • 2 alpha-helices bind the DNA major groove
  • 2 lac l dimers form tetramer
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45
Q

How is the lac repressor formed?

A

Binding of 2 lac l protein dimers

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46
Q

What type of sequence does the lac repressor bind to?

A

Double stranded DNA sequence of operator

Palindromic sequence of 26bp

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47
Q

What occurs in catabolite repression?

A

Involves:

  • cAMP
  • Positive regulator protein (catabolite repressor protein CRP)

A dimer of CRP is activated by a single cAMP
cAMP controlled by levels of glucose
low glucose = more cAMP
CRP interacts with C-terminal domain of the alpha subunit of RNAP to activate it

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48
Q

Where is the TATA box found, what is the sequence and what does it do?

A
  • ~ 25-35 bp upstream of start site
  • TATA(AT)A
  • Used to position RNAP II for transcription initiation
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49
Q

What nucleotides at what positions are characteristic of an initiator?

A

C at -1, A at +1 (transcription start site)

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50
Q

What are the 2 core promoters?

A

TATA box and initiator

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51
Q

What is the proximal control element?

A

CpG islands

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52
Q

What are the 3 distal control elements?

A

Enhancers, Silencers and Insulators

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53
Q

How do genes that have no TATA box or Initiator initiate transcription?

A

Using CpG islands

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54
Q

What are CpG islands and where are they found?

A

CG-rich stretches of DNA, 20-50 nt long, within ~100 bp upstream of start site

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55
Q

Typically how far upstream of the start site are proximal control elements?

A

100-200 bp

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56
Q

Typically how far upstream of the start site are distal control elements?

A

More than 200 bp

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57
Q

Where are the 5 places that enhancers can be found?

A 
50 or more kilobases from promoter they control
upstream of promoter
downstream of promoter
within an intron
downstream from final exon of gene
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58
Q

How do enhancers and silencers work?

A

Binding of factors to enhancers and silencers results in DNA looping allowing interaction with RNA Pol II

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59
Q

What are the 2 types of insulators called?

A

Barrier insulator

Enhancer blocking insulator

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60
Q

What do barrier insulators do?

A

Lie on the border of eu- and heterochromatin domains and safeguard agaisnt the spread of heterochromatin, and thus the chromatin-mediated silencing

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61
Q

What do enhancer blocking barriers do?

A

Protect against gene activation by enhancers and interfere with enhancer-promoter interaction only is insulator is located between enhancer + promoter

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62
Q

What is transcription in eukaryotes carried out by?

A

RNA pol II and general transcription factors (GTFs)

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63
Q

What 5 things are GTFs involved in?

A
  • Promoter recognition
  • RNAP recruitment
  • Interaction with regulatory factors
  • DNA unwinding
  • Transcription start site (TSS) recognition
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64
Q

Describe the process of transcription initiation

A

1) TFIID (which is a GTF) binds to the TATA box
- TFIID consists of:
> TATA binding protein which binds to TATAA
> 10-12 other polypeptides called TBP-associated factors

2) TBP binds to TFIIB forming a TBP-TFIIB complex

3) TFIIB acts as bridge to RNAPII
RNAPII binds to complex associated with TFIIF
TFIIE and TFIIH are then required to bind for initiation of transcription

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65
Q

What are the 2 important roles of TFIIH?

A

Helicase activity that unwinds DNA

Protein kinase activity that causes RNA Pol II phosphorylation

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66
Q

How many subunits does RNAP II have?

A

12

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67
Q

What are the subunits in the core domain and what do they do?

A

Rpb1 - binds DNA
Rpb2 - binds dNTP
Rpb3 + 11 - assembly factors

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68
Q

Where is the carboxy-terminal domain (CTD) of RNAPII found?

A

In the Rpb1 subunit

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69
Q

What is the structure of the CTD?

A

Multiple tandem heptapeptides with repeating sequence

Tyr - Ser - Pro - Thr - Ser - Pro - Ser

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70
Q

How many repeats does mammalian CTD have?

A

52

71
Q

What is the CTD code?

A

combination of different CTD modifications (eg phosphorylation) that allows recruitment and interaction with RNAP II

72
Q

What 3 roles does Ser 5 have?

A
  • Phosphorylation of Ser5 by TFIIH marks initiation
  • Ser5 P marks the 5’ of gene
  • Ser5 P recruits enzymes to cap 5’ end
73
Q

What molecule phosphorylates Ser2 and what is the result?

A
  • Elongation factor PTEF responsible for modification

- Ser2 P converts RNAPII to elongating form (elongation occurs)

74
Q

How are Ser2 and Ser5 involved in termination?

A

Dephosphorylation linked to termination

75
Q

What sequence in the 3’ region signals for cleavage to create a 3’ end?

A

AAUAAA

76
Q

What are the 3 components of the protein complex needed for polyadenylation?

A

Specificity factor
Endonuclease
Poly(A) polymerase

77
Q

How many A residues are added in polyadenylation?

A

approx 200

78
Q

What are the 2 cleavage models involved in termination?

A

Allosteric model - cleavage + polyadenylation cause conformational change in RNA Pol II, pausing it

Torpedo model - Exonuclease binds to 5’ of RNA after cleavage. Catches up with RNA Pol II causing transcription to terminate

79
Q

What 2 domains do all eukaryotic transcription factors have and what do they do?

A

DNA binding domain - binds DNA

Activator/Repressor domain - interact with other proteins to activate/block transcription

80
Q

What is a homeobox and what is its function?

A

DNA binding domain of homeodomain proteins

Important role in animal development

81
Q

What are the 4 stages of Drosophila development?

A

1) Ferilisation
2) Larva
3) Pupae
4) Adult

82
Q

What is a zinc finger?

A

Domain that folds around a central Zn2+ ion

83
Q

Multiple zinc fingers make up the DNA binding domain and bind to which area of DNA?

A

Major groove

84
Q

What is the function and structure of leucine zippers?

A
  • DNA binding domain

- Have hydrophobic leucine at every 7th position in the C-terminal portion of their binding domains

85
Q

Why are the leucine residues in leucine zippers important?

A

Allow dimerisation

Protein dimers bind to DNA

86
Q

What are oncogenes?

A

Genes that have a potential to cause cancer

87
Q

How are leucine zippers involved with oncogenes Jun + Fos?

A

Jun and Fos form dimers via leucine zippers

Dimer binds to genes containing the DNA response element TRE (TGAG)

88
Q

How are helix-loop-helix’s (DNA binding domain) formed?

A

Formed by a small helix and a large helix

89
Q

What are the roles of the small and large helix in helix-loop-helix’s?

A
  • Small helix important for dimerisation

- Large helix binds DNA motif called E-box

90
Q

What is an activation domain?

A

A protein domain that activates transcription when it is fused to a DNA binding domain

91
Q

What are the 2 types of activation domain and describe each

A

Acidic activation domain
- rich is acidic amino acids (Glutamate, glutamine, aspartate, asparagine)

Glutamine rich activation domains
- found in drosophila and mammalia transcription factors

92
Q

What are the 2 main types of amino acid sequence that can be repression domains?

A

1) approx 20 amino acids long and containing high proportions of hydrophobic residues (e.g glycine, alanine, valine)
2) Contain high proportions of basic residues (e.g histidine, lysine, arginine)

93
Q

What is Wilms’s tumor and what is it caused by?

A
  • Kidney tumors in early life

- Mutations in WT1 gene

94
Q

What is the structure and function of WT1 protein?

A
  • Has a zinc finger DNA binding domain
  • Has a repression domain
  • In healthy individuals, binds to control region of gene encoding a transcription activator called EGR-1
95
Q

What structures are methylated in eukaryotic transcriptional control and what is the result of the methylation?

A
  • CpG islands are methylated

- Methylation associated with gene repression

96
Q

What are the 2 types of methyltransferases and what do they do?

A

1) De novo DNA methyltransferase
- acts on unmethylated DNA

2) Maintenance DNA methylthranserase
- Recognises and acts on hemimethylated sites

97
Q

What are MeCP’s and what do they do?

A
  • Proteins called methyl CpG binding protein
  • bind to methylated CpG islands
  • repress transcription by recruiting repressor and/or limiting RNAPII binding to DNA
98
Q

What is Rett syndrome, what is its prevelence and what are the symptoms?

A
  • Brain disorder in girls
  • 1 in 10,000
  • development of sterotypic hand movements
  • loss of speech
  • loss of ability to walk
  • breathing problems
  • anxiety
99
Q

What causes Rett syndrome?

A

Mutations in MeCP2 gene that codes for MeCP

- Gene expression on when it should be off

100
Q

How long in the full amount of DNA found in each cell?

A

2 m

101
Q

What is a histone composed of?

A

> 2 H2A-H2B dimers

> 1 H3-H4 tetramers (made from 2 H3-H4 dimers)

102
Q

How many turns of DNA does one histone wrap?

A

1.7 turns

103
Q

What is a nucleosome?

A

Histone and associated DNA

104
Q

What is the beads on a string conformaton?

A

Nucleosomes connected together by histone H1 and linker DNA

105
Q

In what 3 ways is chromatin rgulated?

A

1) Nucleosome positioning/remodeling
2) Histone variants
3) Histone modifications

106
Q

What enzyme is involved in histone positioning and how does it work?

A

Nucleosome Remodeling Complexes (NRC)

Uses ATP to slide nucleosomes along DNA to expose or hide sequences

107
Q

Give an example of a NRC

A

SWI/SNF

108
Q

What are the 2 main histone variants and what are their functions?

A

1) H3.3 Marks actively transcribed genes

2) H2Az Marks promoters

109
Q

In what 2 ways do histone modifications control transcription?

A

1) Changing the charge of the nucleosome to change DNA-histone interactions to make the DNA more or less accessible
2) Allowing binding of specific proteins (effectors) to modified histone tails

110
Q

What is the consequence of acetylation of histones?

A

Removes positive charge
Decreases binding of negative DNA
Heterochromatin —> Euchromatin

111
Q

In relation to the histone code, What are writers, erasers and readers and give an example of each

A

Writers and Erasers modify chromatin
Readers are proteins that bind to chromatin only if a specific amino acid is modified
Writers - acetylases, methylases
Erasers - deacetylases, demethylases
Readers - Bromodomain, Chronodomain, PHD finger

112
Q

What does methylation of K9 on histone H3 cause?

A

Represses transcription

113
Q

What are heterochromatin and euchromatin?

A

Heterochromatin

  • closed chromatin form
  • repression
  • assembles on gene poor regions of genome

Euchromatin - opposite

114
Q

What is the structure and function of the GAL system?

A
  • 2 Genes - GAL10 and GAL1
  • central control regions: UAS - acts as enhancer

Used in the conversion of galactose to glucose for glycolysis

115
Q

How does Gal4 activate transcription of the GAL genes?

A

Gal4 binds to UAS

116
Q

What occurs in both the presence and absence of galactose?

A

No galactose:
Gal80 represses Gal4

Galactose present:

  • Galactose binds to Gal3 causing conformational change
  • Gal 3 binds Gal80
  • Gal3/Gal80 complex cannot bind Gal4
  • Transcription activation
117
Q

What percentage of the human genome is exons?

A

1%

118
Q

What are the 4 origins of long non coding RNA?

A

1) Mutations of protein coding gene
- frameshift that alters ORF but maintains RNA transcript
2) Chromosomal rearrangment
3) Duplications
4) Transposon insertion

119
Q

Explain the terms sense overlapping, antisense RNAs, LincRNA and Intonic RNA

A

Sense overlapping:
the lncRNA sequence overlaps with the sense strand of a protein coding gene
Antisense RNAs:
lncRNA sequence overlaps with the antisense strand
LincRNA:
lncRNA sequence is not located near any other protein coding loci
IntronicRNA:
lncRNA sequence is derived entirely from within an intron

120
Q

What is dosage compensation?

A

Mechanisms employed to compensate for the discrepancy between the presence of 2 X chromosomes in 1 sex and 1 in the other

121
Q

How is X inactivation controlled in mammals?

A
  • X inactivation center (Xic) produces lncRNA called X-inactive-specific transcript (Xist)
  • Xist coats chromosome from which it was transcribed and silences the X chromosome through Xist mediated recruitment of chromatin modifying compleses
  • Tsix regulates Xist transcription
122
Q

Typically how long are sncRNAs and what is their main function?

A

21-24 nt long

gene silencing

123
Q

How is RNA silencing controlled by dicers and argonautes?

A

Double stranded RNA (dsRNA) is processed by dicer or dicer-like proteins into short RNA duplexes
Small RNAs associate with Argonaute proteins to confer silencing

124
Q

What is a karyotype?

A

Chromosome content of a cell or organism

125
Q

What is a Giesma stain and how does it work?

A
  • Used to stain chromosomes in karyotyping

- binds heterochromatin regions rich in A-T base pairs

126
Q

What is the name of a more modern staining technique?

A

Human chromosome painting
Uses chromosome specific fluorescent probe
Fluorescent in situ hybridisation (FISH)

127
Q

What is a gene map?

A

Order of genetic markers on a given chromosome

128
Q

How is genetic distance measured?

A

Frequency of recombination between 2 linked markers

129
Q

What is gene linkage?

A

Genetic markers on same chromosome

130
Q

What units are used in genetic mappng?

A

centiMorgans (cM)

131
Q

What are genetic markers?

A
  • Naturally occuring mutant alleles of genes e.g eye colour gene
  • Requires a phenotype associated in order to track
132
Q

What are the 3 types of physical marker used in gene mapping?

A

1) single nucleotide polymorphisms (SNPs)
2) Variable number tandem repeats (VNTRs)
3) Microsatellites

133
Q

How are SNPs detected?

A

Using restriction fragment length polymorphisms (RFLPs)

134
Q

How are VNTRs used in gene mapping?

A
  • Number of copies vary between individuals
  • Difference between number of repeats detected by restiction fragments and southern blot
  • Repeat units inherited as single unit (physical gene marker)
135
Q

What is the problem with gene mapping?

A

Gives gene framework but doesn’t give info on sequences

136
Q

What 5 things are needed for the dideoxy chain termination method?

A

1) ssDNA template
2) complementary oligonucleotide primer
3) DNA polymerase
4) dNTPs
5) ddNTPs (small amount)

137
Q

What is the basic dideoxy chain termination method procedure?

A

1) Synthesis 15-20nt primer
2) Set up 4 rections
A: dCTP, dGTP, dTTP, dATP/ddATP
etc
3) Use 32p-labelled nucleotides
4) Seperate fragments by PAGE
5) Detect result using autoradiograph

138
Q

How is the dideoxy chain termination method carried out in the modern day?

A
  • Different coloured dyes for each terminator base
  • Load reaction sample into capillary electrophoresis tube
  • Fragments seperate by size
  • Laser and detector detect dyed terminator
  • Computer reads results
139
Q

What is the 2 step strategy for sequencing large genomes?

A

1) Generate overlapping clone of genomic DNA by partial restriction digestion
2) Sequence each fragment and align fagments to generate contigs (overlapping clones that form physical map of genome)
- requires computing

140
Q

What is the name of the next generation sequencing technique?

A

454 sequencing

141
Q

What is genome annotation?

A

Systematic identification in a genome sequence of:

  • protein coding info
  • RNA coding info
142
Q

How is gene annotation performed in simple eukaryotes and bacteria?

A

Computer method to search for ORFs

143
Q

How is gene annotaton performed in complex eukaryotes?

A

Comparing cDNA to genomic DNA to find intron locations

144
Q

How is cDNA made?

A

1) mRNA (fully processed)
2) Hybridise with poly(T) primer
3) Make DNA copy with reverse transcriptase
4) Degrade RNA with RNase H
5) Synthesise new DNA strand using DNA polymerase

145
Q

What is the aim of functional genomics?

A

To understand the relationship between an organisms genome and its phenotype

146
Q

What 3 experimental approaches are used in functional genomics?

A
  • DNA sequencing
  • Transcriptomics
  • Proteomics
147
Q

What is a null allele?

A

Mutant copy of gene that completely lacks its normal function
OR
Deletion of gene from genome

148
Q

How is the 5’ cap formed?

A
  • Terminal phosphate group removed by RNA triphoshatase, leaving bisphosphate group
  • GTP is added to bisphosphate by mRNA guanylytransferase, results in 5’-5’ triphosphate linkage
  • The 7-nitrogen of guanine is methylated by mRNA methyltransferase
149
Q

What are the main 2 functions of the 5’ cap?

A
  • Protects mRNA from degradation
  • Recruitment of translation initiation factors
    PROTECT and SIGNAL
150
Q

What sequences are recgonised for splicing to occur?

A

GU at 5’ splice site

AG at 3’ splice site

151
Q

What sequences are recognised in minor intron splicing?

A

AU-AC

152
Q

How is a free lariat formed?

A
  • 5’ splice site cleaved
  • 5’ end joined to 2’ position at an A in the branch site
  • 3’ splice site cleaved
153
Q

What are snRNPs?

A

small nuclear RNA and proteins involved in splicing

154
Q

What is the sliceosome composed of?

A

snRNPs (U1, U2, U5, U4 and U6) and other proteins

155
Q

What is the first step of splicing?

A

Binding of U1 snRNP to 5’ splice site

156
Q

What is U1 snRNP composed of?

A
  • core Sm proteins
  • 3 U1-specific proteins
  • U1 snRNA
157
Q

What is the E cpmplex?

A

U1 snRNP bound to 5’ splice site
U2AF65 and U2A535 bound to pyrimidine tract between branch site and 3’ splice site
Branch point binding protein binds to branch point
Bound SR proteins

158
Q

What are the 6 stages in pre-mRNA splicing and explain each

A

1) E complex
2) A complex
- ATP dependent step - U2 snRNP binding to branch point, involving U2 RNA binding to form presliceosome or A complex
3) B1 complex
- Trimer containing U5 and U4/U6 snRNAps bind to A complex
- Has all components for splicing
4) B2 complex
- converted to B2 when U1 is released
- allows U6 to allign properly
- U4 dissociates from U6, U6 binds to U2 to form active site
5) C1 complex
First step of transesterification
5’ splice site cleaved, lariat formed
6) C2 complex
3’ splice site cleaved, exons ligated

159
Q

Which groups of introns remove themselves by autosplicing?

A

Group 1 and 2

160
Q

What is required for a group 1 splicing reaction and what is the product?

A

Guanine nucleotide with free 3’OH group required as co factor
Intron released as linear structure

161
Q

How does the size of group 2 introns compare to group 1?

A

larger

162
Q

What is the exon junction complex?

A

A protein complex that assembles at the exon-exon junction during splicing to assist in RNA transport, localisation and degradation

163
Q

What does the EJC bind to to facilitate mRNA export from the nucleus?

A

TAP/Mex

164
Q

What is alternative splicing used for?

A

To generate multiple outputs from a single gene

165
Q

What disease is a result of defects in alternative splicing?

A

Muscular Dystrophy

166
Q

What are Nova and Fox?

A

RNA binding proteins that can promote or silence splice site selection

167
Q

When do Nova and Fox cause suppression of the exon and when do they enhance it?

A
  • Binding upstream of the alternatively spliced exon causes supression
  • Binding downstream enhances
168
Q

What motif do the FOX proteins recognise?

A

UGCAUG

169
Q

What signal causes cleavage to produce a 3’ end?

A

AAUAAA

170
Q

Describe how cleavage and polyadenylation occurs to the 3’ end

A

Cleavage and polyadenylation specific factor (CPSF) binds to AAUAAA and to CSTF which together cuts the RNA
polyA polymerase adds 10 A residues which allows recruitment of polyA binding protein (PABP) which makes poly A tail (around 200 As)

171
Q

What polymerase transcribes pre tRNAs?

A

RNA polymerase III

172
Q

What 3 enzymes are involved in pre-tRNA splicing?

A

1) An endonulease that recognises (Sen54) and cleaves (Sen34/Sen2) the pre tRNA at both ends of the intron
2) Ligase to fuse exons (Trl1)
3) A 2’phosphotransferase that removes the 2’phosphate and spliced tRNA
Reactions are ATP dependant

173
Q

What does the endonuclease consist of and how does it cleave pre-tRNA?

A
  • Yeast endonuclease is a heterotetrameric protein of 2 catalytic subunits (Sen34 and Sen2) and 2 structural subunits (Sen54 and Sen15)
  • Sen54 determines cleavage site
  • Sen34/Sen2 performs endonuclease reaction
  • This generates 2’-3’ cyclic phosphate and 5’-OH termini
174
Q

What are the 3 steps in Trl1 reaction?

A

1) Cyclic phophodiesterase activity opens the cyclic phosphate group to generate 2 phosphate termini
2) Kinase activity to introduce phosphate at 5’-OH termini
3) Ligase activity joins 2 ends

Biomed (18 decks)

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