Gene expression and its control BI501 Flashcards
Name the 3 characteristics of mRNA that make it different to DNA
1) single stranded
2) ribose sugar instead of deoxyribose
3) Uracil replaces Thymine
That is the name of the region that regulates transcription?
Promoter region
What are the 4 stages of transcription?
1) Template recognition
2) Initiation of transcription (Steps 1&2 are INITIATION)
3) ELONGATION
4) TERMINATION
Name 3 characteristics of prokaryotes
Single celled
lack membrane bound organelles
divided into bacteria and archea
Name 3 differences between gene expression if prokaryotes and eukaryotes
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
What are operons and how are they regulated?
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
How are proteins produced using operons?
Genes are transcribed into a single RNA (polycistronic) that is translated into different proteins
What 2 things does transcription require?
1) cis-acting elements (promoter)
2) trans-acting elements (RNAP and transcription factors)
Where are bacterial promoters typically located?
Upstream or at the 5’ end of the transcription initiation site
The promoter sequence defines both the direction of transcription and which strand will be transcribed. What is this strand known as?
Sense strand
What are the 5 motifs in prokaryotic promoters and what part of the RNAP recognises each?
> 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)
What does the RNAP holoenzyme consist of?
Core enzyme and the sigma factor
What is the core enzyme made up of?
- 2 alpha subunits
- 2 beta subunits
- 1 omega subunit
What are the functions of the beta subunits?
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
What are the functions of the dimer formed by 2 alpha subunits?
- serves as scaffold for assembly of core enzyme
- contribute to promoter recognition
- bind some activators
What is the function of the omega subunit?
Plays a role in enzyme assembly
What are the 2 most important functions of the sigma factors?
1) Directing the catalytic core of the RNAP to the appropriate transcription start sites
2) Suppressing Nonspecific Transcription Initiation
Which sigma factor domains have DNA binding elements and which prokaryotic binding motif do they bind to?
- sigma4 binds the -35 motif
- sigma3 binds the extended -10 motif
- sigma2 binds to the -10 and discriminator motifs
Can free sigma factors bind to DNA promoters?
No
Sigma factors must be coupled with the core enzyme in order to interact with the promoter
What are the 2 mechanisms that contribute to suppressing the DNA-binding capacity of free sigma factors?
1) Conformational restriction
2) Auto-inhibitory restriction
In what 2 ways do sigma factors suppress nonspecific transcription initiation?
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
What is the transcription bubble?
RNAP unwinds 13 bp of DNA after it locates a promoter through recognition of DNA motifs
What occurs after a transcription bubble is formed?
Nucleotides are incorporated into RNA and a ternary complex is formed (complex containing RNA, DNA and the enzyme)
How are oligonucleotides produced and typically how long are they?
Produced by repeated cycles of abortive initiation
approx 20nt long
At what point is the sigma factor released from the RNAP?
When the RNA chain reaches approx 10 bases long
What are the 2 types of termination?
1) Intrinsic termination
2) Rho-dependent termination
Describe intrinsic termination
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
Describe Rho-dependant termination
- 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
Name the 3 stages in which gene expression can be controlled?
1) Transcription
2) RNA processing
3) Translation
What effect will both an activator and a suppressor have when bound to the operator?
Activator - Initiate transcription
Repressor - Repress transcription
What is positive regulation?
When the binding of an activator is a necessary prerequisite for transcription to initiate
What is negative regulation?
When the prevention binding of an activator is a necessary prerequisite for transcription to initiate
How does an activator initiate transcription (1 way) and how does a repressor repress transcription (2 ways)
Activator - helps tether RNAP to promoter
Repressor - physically interfering with RNAP binding
- Impeding elongation
What 2 states can activators and repressors exist in?
- DNA binding
- Non DNA binding
How do activators and repressors achieve 2 states?
Binding of allosteric effectors (inducers and co-repressors) to allosteric sites on activator/repressor
How is transcription initiation regulated in prokaryotes?
Competition of sigma factors - different sigma factors activated by different mechanisms
What is the lack operon?
Cluster of genes required for metabolism of lactose
When is the lac operon on and off?
ON in presence of lactose
OFF in presence of glucose
What are the 3 genes in the lac operon and what does each do?
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
What gene codes for the lac repressor?
lac l gene
What does the lac repressor do?
Represses lacZYA transcription
What are the 2 recognition sites on the lac repressor and what do they bind to?
- One that binds to DNA
- Allosteric site that binds the lactose allosteric effector (allolactose)
What occurs when allolactose is present?
Binds to allosteric site on lac repressor, causes conformational change that inactivates DNA binding activity of repressor
lacZYA transcription can occur
What is the structure of the lac l protein?
- DNA binding domain contains a helix-turn-helix motif
- 2 alpha-helices bind the DNA major groove
- 2 lac l dimers form tetramer
How is the lac repressor formed?
Binding of 2 lac l protein dimers
What type of sequence does the lac repressor bind to?
Double stranded DNA sequence of operator
Palindromic sequence of 26bp
What occurs in catabolite repression?
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
Where is the TATA box found, what is the sequence and what does it do?
- ~ 25-35 bp upstream of start site
- TATA(AT)A
- Used to position RNAP II for transcription initiation
What nucleotides at what positions are characteristic of an initiator?
C at -1, A at +1 (transcription start site)
What are the 2 core promoters?
TATA box and initiator
What is the proximal control element?
CpG islands
What are the 3 distal control elements?
Enhancers, Silencers and Insulators
How do genes that have no TATA box or Initiator initiate transcription?
Using CpG islands
What are CpG islands and where are they found?
CG-rich stretches of DNA, 20-50 nt long, within ~100 bp upstream of start site
Typically how far upstream of the start site are proximal control elements?
100-200 bp
Typically how far upstream of the start site are distal control elements?
More than 200 bp
Where are the 5 places that enhancers can be found?
50 or more kilobases from promoter they control upstream of promoter downstream of promoter within an intron downstream from final exon of gene
How do enhancers and silencers work?
Binding of factors to enhancers and silencers results in DNA looping allowing interaction with RNA Pol II
What are the 2 types of insulators called?
Barrier insulator
Enhancer blocking insulator
What do barrier insulators do?
Lie on the border of eu- and heterochromatin domains and safeguard agaisnt the spread of heterochromatin, and thus the chromatin-mediated silencing
What do enhancer blocking barriers do?
Protect against gene activation by enhancers and interfere with enhancer-promoter interaction only is insulator is located between enhancer + promoter
What is transcription in eukaryotes carried out by?
RNA pol II and general transcription factors (GTFs)
What 5 things are GTFs involved in?
- Promoter recognition
- RNAP recruitment
- Interaction with regulatory factors
- DNA unwinding
- Transcription start site (TSS) recognition
Describe the process of transcription initiation
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
What are the 2 important roles of TFIIH?
Helicase activity that unwinds DNA
Protein kinase activity that causes RNA Pol II phosphorylation
How many subunits does RNAP II have?
12
What are the subunits in the core domain and what do they do?
Rpb1 - binds DNA
Rpb2 - binds dNTP
Rpb3 + 11 - assembly factors
Where is the carboxy-terminal domain (CTD) of RNAPII found?
In the Rpb1 subunit
What is the structure of the CTD?
Multiple tandem heptapeptides with repeating sequence
Tyr - Ser - Pro - Thr - Ser - Pro - Ser
How many repeats does mammalian CTD have?
52
What is the CTD code?
combination of different CTD modifications (eg phosphorylation) that allows recruitment and interaction with RNAP II
What 3 roles does Ser 5 have?
- Phosphorylation of Ser5 by TFIIH marks initiation
- Ser5 P marks the 5’ of gene
- Ser5 P recruits enzymes to cap 5’ end
What molecule phosphorylates Ser2 and what is the result?
- Elongation factor PTEF responsible for modification
- Ser2 P converts RNAPII to elongating form (elongation occurs)
How are Ser2 and Ser5 involved in termination?
Dephosphorylation linked to termination
What sequence in the 3’ region signals for cleavage to create a 3’ end?
AAUAAA
What are the 3 components of the protein complex needed for polyadenylation?
Specificity factor
Endonuclease
Poly(A) polymerase
How many A residues are added in polyadenylation?
approx 200
What are the 2 cleavage models involved in termination?
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
What 2 domains do all eukaryotic transcription factors have and what do they do?
DNA binding domain - binds DNA
Activator/Repressor domain - interact with other proteins to activate/block transcription
What is a homeobox and what is its function?
DNA binding domain of homeodomain proteins
Important role in animal development
What are the 4 stages of Drosophila development?
1) Ferilisation
2) Larva
3) Pupae
4) Adult
What is a zinc finger?
Domain that folds around a central Zn2+ ion
Multiple zinc fingers make up the DNA binding domain and bind to which area of DNA?
Major groove
What is the function and structure of leucine zippers?
- DNA binding domain
- Have hydrophobic leucine at every 7th position in the C-terminal portion of their binding domains
Why are the leucine residues in leucine zippers important?
Allow dimerisation
Protein dimers bind to DNA
What are oncogenes?
Genes that have a potential to cause cancer
How are leucine zippers involved with oncogenes Jun + Fos?
Jun and Fos form dimers via leucine zippers
Dimer binds to genes containing the DNA response element TRE (TGAG)
How are helix-loop-helix’s (DNA binding domain) formed?
Formed by a small helix and a large helix
What are the roles of the small and large helix in helix-loop-helix’s?
- Small helix important for dimerisation
- Large helix binds DNA motif called E-box
What is an activation domain?
A protein domain that activates transcription when it is fused to a DNA binding domain
What are the 2 types of activation domain and describe each
Acidic activation domain
- rich is acidic amino acids (Glutamate, glutamine, aspartate, asparagine)
Glutamine rich activation domains
- found in drosophila and mammalia transcription factors
What are the 2 main types of amino acid sequence that can be repression domains?
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)
What is Wilms’s tumor and what is it caused by?
- Kidney tumors in early life
- Mutations in WT1 gene
What is the structure and function of WT1 protein?
- 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
What structures are methylated in eukaryotic transcriptional control and what is the result of the methylation?
- CpG islands are methylated
- Methylation associated with gene repression
What are the 2 types of methyltransferases and what do they do?
1) De novo DNA methyltransferase
- acts on unmethylated DNA
2) Maintenance DNA methylthranserase
- Recognises and acts on hemimethylated sites
What are MeCP’s and what do they do?
- Proteins called methyl CpG binding protein
- bind to methylated CpG islands
- repress transcription by recruiting repressor and/or limiting RNAPII binding to DNA
What is Rett syndrome, what is its prevelence and what are the symptoms?
- Brain disorder in girls
- 1 in 10,000
- development of sterotypic hand movements
- loss of speech
- loss of ability to walk
- breathing problems
- anxiety
What causes Rett syndrome?
Mutations in MeCP2 gene that codes for MeCP
- Gene expression on when it should be off
How long in the full amount of DNA found in each cell?
2 m
What is a histone composed of?
> 2 H2A-H2B dimers
> 1 H3-H4 tetramers (made from 2 H3-H4 dimers)
How many turns of DNA does one histone wrap?
1.7 turns
What is a nucleosome?
Histone and associated DNA
What is the beads on a string conformaton?
Nucleosomes connected together by histone H1 and linker DNA
In what 3 ways is chromatin rgulated?
1) Nucleosome positioning/remodeling
2) Histone variants
3) Histone modifications
What enzyme is involved in histone positioning and how does it work?
Nucleosome Remodeling Complexes (NRC)
Uses ATP to slide nucleosomes along DNA to expose or hide sequences
Give an example of a NRC
SWI/SNF
What are the 2 main histone variants and what are their functions?
1) H3.3 Marks actively transcribed genes
2) H2Az Marks promoters
In what 2 ways do histone modifications control transcription?
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
What is the consequence of acetylation of histones?
Removes positive charge
Decreases binding of negative DNA
Heterochromatin —> Euchromatin
In relation to the histone code, What are writers, erasers and readers and give an example of each
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
What does methylation of K9 on histone H3 cause?
Represses transcription
What are heterochromatin and euchromatin?
Heterochromatin
- closed chromatin form
- repression
- assembles on gene poor regions of genome
Euchromatin - opposite
What is the structure and function of the GAL system?
- 2 Genes - GAL10 and GAL1
- central control regions: UAS - acts as enhancer
Used in the conversion of galactose to glucose for glycolysis
How does Gal4 activate transcription of the GAL genes?
Gal4 binds to UAS
What occurs in both the presence and absence of galactose?
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
What percentage of the human genome is exons?
1%
What are the 4 origins of long non coding RNA?
1) Mutations of protein coding gene
- frameshift that alters ORF but maintains RNA transcript
2) Chromosomal rearrangment
3) Duplications
4) Transposon insertion
Explain the terms sense overlapping, antisense RNAs, LincRNA and Intonic RNA
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
What is dosage compensation?
Mechanisms employed to compensate for the discrepancy between the presence of 2 X chromosomes in 1 sex and 1 in the other
How is X inactivation controlled in mammals?
- 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
Typically how long are sncRNAs and what is their main function?
21-24 nt long
gene silencing
How is RNA silencing controlled by dicers and argonautes?
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
What is a karyotype?
Chromosome content of a cell or organism
What is a Giesma stain and how does it work?
- Used to stain chromosomes in karyotyping
- binds heterochromatin regions rich in A-T base pairs
What is the name of a more modern staining technique?
Human chromosome painting
Uses chromosome specific fluorescent probe
Fluorescent in situ hybridisation (FISH)
What is a gene map?
Order of genetic markers on a given chromosome
How is genetic distance measured?
Frequency of recombination between 2 linked markers
What is gene linkage?
Genetic markers on same chromosome
What units are used in genetic mappng?
centiMorgans (cM)
What are genetic markers?
- Naturally occuring mutant alleles of genes e.g eye colour gene
- Requires a phenotype associated in order to track
What are the 3 types of physical marker used in gene mapping?
1) single nucleotide polymorphisms (SNPs)
2) Variable number tandem repeats (VNTRs)
3) Microsatellites
How are SNPs detected?
Using restriction fragment length polymorphisms (RFLPs)
How are VNTRs used in gene mapping?
- 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)
What is the problem with gene mapping?
Gives gene framework but doesn’t give info on sequences
What 5 things are needed for the dideoxy chain termination method?
1) ssDNA template
2) complementary oligonucleotide primer
3) DNA polymerase
4) dNTPs
5) ddNTPs (small amount)
What is the basic dideoxy chain termination method procedure?
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
How is the dideoxy chain termination method carried out in the modern day?
- 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
What is the 2 step strategy for sequencing large genomes?
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
What is the name of the next generation sequencing technique?
454 sequencing
What is genome annotation?
Systematic identification in a genome sequence of:
- protein coding info
- RNA coding info
How is gene annotation performed in simple eukaryotes and bacteria?
Computer method to search for ORFs
How is gene annotaton performed in complex eukaryotes?
Comparing cDNA to genomic DNA to find intron locations
How is cDNA made?
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
What is the aim of functional genomics?
To understand the relationship between an organisms genome and its phenotype
What 3 experimental approaches are used in functional genomics?
- DNA sequencing
- Transcriptomics
- Proteomics
What is a null allele?
Mutant copy of gene that completely lacks its normal function
OR
Deletion of gene from genome
How is the 5’ cap formed?
- 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
What are the main 2 functions of the 5’ cap?
- Protects mRNA from degradation
- Recruitment of translation initiation factors
PROTECT and SIGNAL
What sequences are recgonised for splicing to occur?
GU at 5’ splice site
AG at 3’ splice site
What sequences are recognised in minor intron splicing?
AU-AC
How is a free lariat formed?
- 5’ splice site cleaved
- 5’ end joined to 2’ position at an A in the branch site
- 3’ splice site cleaved
What are snRNPs?
small nuclear RNA and proteins involved in splicing
What is the sliceosome composed of?
snRNPs (U1, U2, U5, U4 and U6) and other proteins
What is the first step of splicing?
Binding of U1 snRNP to 5’ splice site
What is U1 snRNP composed of?
- core Sm proteins
- 3 U1-specific proteins
- U1 snRNA
What is the E cpmplex?
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
What are the 6 stages in pre-mRNA splicing and explain each
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
Which groups of introns remove themselves by autosplicing?
Group 1 and 2
What is required for a group 1 splicing reaction and what is the product?
Guanine nucleotide with free 3’OH group required as co factor
Intron released as linear structure
How does the size of group 2 introns compare to group 1?
larger
What is the exon junction complex?
A protein complex that assembles at the exon-exon junction during splicing to assist in RNA transport, localisation and degradation
What does the EJC bind to to facilitate mRNA export from the nucleus?
TAP/Mex
What is alternative splicing used for?
To generate multiple outputs from a single gene
What disease is a result of defects in alternative splicing?
Muscular Dystrophy
What are Nova and Fox?
RNA binding proteins that can promote or silence splice site selection
When do Nova and Fox cause suppression of the exon and when do they enhance it?
- Binding upstream of the alternatively spliced exon causes supression
- Binding downstream enhances
What motif do the FOX proteins recognise?
UGCAUG
What signal causes cleavage to produce a 3’ end?
AAUAAA
Describe how cleavage and polyadenylation occurs to the 3’ end
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)
What polymerase transcribes pre tRNAs?
RNA polymerase III
What 3 enzymes are involved in pre-tRNA splicing?
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
What does the endonuclease consist of and how does it cleave pre-tRNA?
- 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
What are the 3 steps in Trl1 reaction?
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
