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