Augustus Barbosa Pretranscriptional control Flashcards
What is the difference between pre-transcriptional control and post-transcriptional control?
Which of these is most important in Prokaryotes and why?
Pre-transcriptional control is regulated at the initiation of transcription of mRNA transcript
Post-transcriptional control occurs after the RNA transcript has been made, and prevents trnaslation of the transcript
Pre-transcriptional control is most important in prokaryotes as transcription and translation can occur simultaneously, this results in a loss of regulation, but faster ability to produce proteins which is important for prokaryotes as they live in highly dynamic environments and have short lifespans
What are subunits of the prokaryote RNA polmerase and what is there function?
There are two of each domain
Alpha unit interacts with sigma factors to initiate transcription
Beta unit which produces RNA
Omega unit which stabalizes the enzyme
What are sigma factors?
Proteins which form a link between DNA and RNA,
Different sigma factors recognise different promotors allowing for gene regulation
e.g.
Sigma 70= Housekeeping genes
How is the lac operon regulated?
The lac operon contains genes for the enzyme beta galactosidase,, permease and transacetylase which are ll the enzymes required for lactose metabolism,
As the lac operon is a negative control operon, there is a repressor protein which blocks the binding of sigma 70 in the ground state (absence of lactose), if lactose is present this binds to the repressor inactivating it allowing the binding of sigma 70 and therefore gene transcription.
The Lac operon is unusual as it also demonstrates positive control, as there is a CAP site upstream of the promoter which is activated by high levels of cAMP in low glucose environments activation of the CAP protein leads to it binding to the CAP site and drives sigma 70 binding
How is the tryptophan operon regulated?
High levels of tryptophan will result in tryptophan binding to the repressor protein activating it in an example of negative control
Tryptophan operon is also regulated by attenuation, as a result of cross talk between the ribosome and RNA polymerase, if the ribosomes are fast then one hair pin loop forms with a termination sequence preventing translation
If the ribosomes are slow then two hair pin loops form disrupting the termination signal allowing translation to continue
What are the RNA polymerases in eukaryotes and how are they controlled?
RNA pol. 1= Uncontrolled
RNA pol 2.= Control via the carboxy terminal tail, phosphorylation can be used to regulate enzymatic activity, low levels or no phosphorylation allows for the initiation of transcrition but some phosphorylation is required for elongation and mRNA cap
formation
This results in a need for more proteins to be recruited to attract RNA pol.2 as it is a weakly binding enzyme
RNA pol.3= unregulated
How is pretranscriptional control found in eukaryotes via chromatin?
Densly packed chromatin prevents RNA pol. binding, therefore eukaryotes must exert positive control to unwind sections of chromatin where essential genes are located
Chromatin plays a role in gene silencing seen in cell differentiation and embryogenesis, X chromosome inactivation and epigenetic inheritance
Condensation of chromatin is driven by acetylation, methylation, phosphorylation and ubiquitation, this can provide distinct chemical signals allowing for proteins recognition
How is RNA pol.2 attracted to the DNA to initiate transcription?
All eukaryote genes have a core promoter which is required for formation of the pre-initiation complex
This is recognised by TATA Box binding protein which then binds TFIIB to allow connection to the DNA, TFIIF and RNA pol. II are recruited and placed over the transcription start site, TFIIE and TFIIH dissociate, TFIIH acts as a helicase and phosphorylates RNA pol. II to release the capping complex
The location of the core promoter must be conserved, some genes will also use other controlling structures such as proximal promoters which are more prone to mutations
What is the histone Code?
Chemical modifications on the histone tail causing specific, recognisable sequences
Chromodomain is a region recognised after the specific lysines are methylated
Bromodomain is a region recognised after specific lysines are acetylated
HP1 binds to chromodomains revealing chromoshadow domains, which promotes oligomerisation of HP1 wtih itself casuing chromatin compaction and gene silencing
What is the difference between methylation and acetylation with regards to chromatin?
Methyl groups are stable allowing them to last a long time playing a key role in cellular development
Acetylgroups however take only a short time to turn over resulting in constant re-establishment of the euchromatin being neccessary
Modifiction occurs via both activating acetylation activity or deactivating deacetylation activity
This modification occurs via double domained repressor/activator proteins
How does initiation of transcription occur in eukaryotes?
The chromatin is modulated via chemical modification of the histone SW1/SNF complex allowing for decondensation and DNA accessibility of the DNA Large mediator (60-80 proteins) with acetylase activity bridges the gap between the promoter sequence and the enhancer, only one mediator is needed due to the large number of subunits The preinitiation complex is formed as the euchromatin is flexible, therefore it can bend into the required hairpin loop formation and attract transcription factors which can in turn stabalize the DNA to attract RNA pol. II
How does the technique of transcription of genes in cell free extracts work?
The nucleus is purified away from the cytoplasm and other organelles
The salt extract isolates the transcription factors bound to DAN
X-Ray crystallography can then aid in determining the structures of these transcription factors
How does the technique of recombinant DNA work?
DNA cloning allows genes and regulatory elements to be bloned and introduced into a cell
Genetic engineering allows the construction of genetic constructs
Reporter genes are used to determine uptake are used
What is a Gel Mobility Shift Assays?
Radio labelled DNA fragments are mixed with nuclear protein extracts, DNA that binds protein will slow migration in gel, shift is sequence specific so the sequence can be determined through binding the sequence to resin, washing it with nuclear extract, this traps the nuclear specific binding protein
What is the technique of DNAse footprinting?
Protein binds to a specifc DNA sequence preventing enzymatic digestion which can be mapped via gel electrophoresis
