Lecture 14 - Regulation of transcription Flashcards
Why do cells need ot regulate transcription?
Prokaryotes an eukaryotes have many genes that encode different proteins. However, although a cell might require lots of some proteins, they might only require a little of others. They also only need to produce some proteins at particular times or only ever need to produce a subset of the proteins encoded by the genome.
What are transcription factors?
- Proteins that regulate transcription
- Contain a DNA binding domain and a transactivating domain
- It has been estimated that 10% of the genes in the human genome code for TFs.
Shapes include - Helix-turn-Helix - Found in all types of organisms.
- Coiled coil domains - Found in leucine zipper and helix-loop-helix motifs.
Zinc finger domains - very common in humans.
Describe the regulatory mechanisms of transcription in prokaryotes.
- RNA polymerase holoenzyme can transcribe any gene with a function promotor
- Targeted gene regulation is used most at:
○ Transcription initiation
○ Elongation or termination
○ Regulation from the transcribed RNA itself - Regulatory proteins that decrease transcription are called repressors
- Proteins that increase transcription are activators
The regulatory DNA sequences they bind to are called operators
- Targeted gene regulation is used most at:
Describe regulation of transcription in Eukaryotes.
Distal (1000s bp) regulatory sequences more frequently used than in bacteria- called enhancers or enhancer elements (can be upstream or downstream of the gene
Regulatory sequences frequently bind several regulatory proteins- allows expression to be tuned.
Describe control of gene expression in bacteria.
Constitutive or ‘housekeeping’ genes - are always expressed and encode proteins that are always needed
Regulated genes - their expression required only under certain circumstances. Their expression is regulated at the level of transcription.
e.g. LexA represses expression of SOS genes in the absence of damage
When replication fork stalls RecA binds to single-stranded DNA and becomes activated to cleave the LexA repressor.
At the damaged replication fork LexA is cleaved and inactivated so it can not bind to the operator this means that there is transcription of SOS genes and production of SOSO proteins including RecA and DinI
Once the DNA damage is repaired RecA is inhibited by DinI and newly synthesised LexA repressor binds to DNA so the SOS gene transcription stops.
What are operons
Expression of genes which encode proteins that work together is co-ordinated by organisation of genes into operons. In an operon genes adjacent to each other are transcribed together into one polycistronic mRNA - which is then translated into separate proteins encoded by each gene present in the operon.
How is the E.coli lac operon used?
E. Coli uses glucose as an energy source
When lactose is added as the sole carbons source there is a rapid increase in lac mRNA transcript causing a rapid synthesis of enzymes required to metabolise lactose. (β-galactosidase converts lactose to galactose and glucose or to allolactose)
The lac operon (encodes genes lacZ, lacY and lacA) is translated to produce a single mRNA that is translated to produce three proteins
* β-galactosidase (lacZ)
* Permease (lacY)
* Transacetylase (LacA)
The lacI gene is situated immediately upstream of the operon - it has its own promoter and is constitutively expressed. It plays an important role in regulating transcription of the lac operon.
When there is no lactose present, LacI binds to the lac operator so no transcription occurs as RNA Pol cant bind.
When lactose is present, allolactose binds to LacI altering its shape this allows transcription to occur (derepression)
The presence of glucose inactivates adenylate cyclase and therefore reduces levels of cAMP (cAMP is therefore an indicator of glucose levels)
cAMP receptor protein (CRP) only binds to DNA in the presence of cAMP. When it binds it bends the DNA 90°. (The Dimerised cap can bind to DNA - when tow monomers of CAP are dimerised around cAMP and can then bind to DNA)
* CAP-cAMP binding upstream of the promotor facilitates RNA polymerase holoenzyme binding
* It activates more than 100 different E. coli promotors when glucose levels are low (including lac genes)
Summary - lac operon is regulated by a combination of negative inducible regulation (Lac repressor) and positive regulation via (cAMP-CAP)
When there is low glucose and lactose is available the gene is strongly on
When there is low glucose and lactose is unavailable gene is off (as lac repressor is bound)
When there is high glucose and lactose is available the gene is weakly on (There is no cAMP-CAP)
When there is high glucose and lactose is unavailable the gene is off.
Describe negative regulation of transcription by repression.
Operons for anabolic (biosynthetic) pathways are turned off when the end product is readily available - repressible regulation
E.g. trp operon
5 genes encoding enzymes for tryptophan biosynthesis are expressed in an operon as a polycistronic mRNA - turned off by excess tryptophan. (the regulatory gene for trp isn’t near the operon)
trpR encodes an aporepressor:
* In an absence of tryptophan no binding of TrpR aporepressor to operator so transcription occurs
* In the presence of excess tryptophan the aporepressor is activated by binding tryptophan. It then binds to the operator preventing transcription.
There is a second regulatory mechanism where the concentration of tryptophan is low:
* Tryptophan starvation - maximal expression of trp genes.
* Tryptophan limitation - less than maximal expression of trp genes
Controls ration of full-length transcripts to short 140 bp transcripts terminated within trpL leader region - attenuation
Mechanism is dependent on translation.
Four regions of the trp leader mRNA can form alternative secondary structures (formation depends on ribosome progression as mRNA is translated)
* In the presence of tryptophan - attenuation at the second regulatory site in response to Trp-tRNA levels terminates transcription before the structural genes are transcribed.
Absence of tryptophan - stalling of ribosome at Trp codons results in antitermination - transcription proceeds to produce full length mRNA encoding biosynthesis enzymes.
What are riboswitches
Riboswitches are portions of a transcript that can directly bind a small molecule that controls the RNA secondary structure, regulating transcription or translation
Riboswitches have two regions
* The Aptamer that binds to the metabolite
An expression platform which controls transcription or translation
Describe the adenine riboswitch.
The B. subtilis adenine riboswitch regulates adenine synthesis and transport. Gene expression depends on whether a terminator or anti-terminator forms
* In low adenine, an RNA structure forms so the Regions 2 and 3 form an anti-terminator and transcription proceeds.
In high adenine regions 3 and 4 form a terminator.
