Week 10 - Gene Expression part 3 Flashcards
Promoters of bacteria
They are sequences upstream from a gene that controls gene expression usually <200 bases
Give an example of a simple eukaryotic promoter
Consists of a silencer and a UAS which are the transcription factor binding site, followed by the TATA box which is the RNA polymerase binding site
S. cerevisiae is an example of a simple promoter
Give an example of a higher eukaryotic promoter
Has upstream Insulators, silencers, and enhancers. Followed by TATA and initiator sequences and then downstream promoter elements
How is primer extension used to determine the transcription start point?
primer anneals to RNA transcript, cDNA is produced with RT and terminates at 5’ end of the mRNA. mRNA is then denatured leaving only the DNA which can be correlated with the DNA sequence.
Can only be used to determine the 5’ end of the mRNA, and as such is used to confirm S1 nuclease mapping
PCR based analysis - rapid amplification of cDNA ends (RACE)
Can be used to map the ends of the transcript
Primer specific to internal region of mRNA used for cDNA creation via RT, once cDNA terminates at 5’ end of transcript terminal transferase adds poly(A) tail. Second primer anneals to poly(A) 2nd strand of cDNA is synthesized, PCR is complete and sequence the product
S1 nuclease mapping
Uses a labelled DNA probe of known size and complimentary to the 5’ region of the gene, Probe is annealed to mRNA, Annealing protects the 5’ end of the DNA probe from S1 digestion, mRNA is degraded by alkali and sized using gel electrophoresis. Compare to DNA and duduce start site
Methods for monitoring DNA to protein interactions
EMSA
DNase protection (footprinting)
ChIP
What is the most important relationship between Protein and DNA
DNA-binding proteins bind to recognition sequences, With proteins often having a binding region much larger than the actual recog sequences
EMSA (Electrophoretic Mobility Shift Assay)
key principle is that bound DNA has a greater mass than unbound DNA = moves slowly through a gel.
Target is usually DNA upstream of a promoter
Specificity of protein can be confirmed using antibodies for a super shift
DNase 1 foot printing
Bound DNA will be protected from DNase 1 nuclease digestion, foot printing provides more precise info on the location of a protein binding site on DNA
How is analysis of DNase 1 foot printing performed?
With polyacrylamide gel electrophoresis and autoradiography, Only the bound DNA that was protected will show up, the size test has indicators of where the proteins were bound and the size of fragments down either side.
Deletion analysis
DNA Chromatin Immunoprecipitation (ChIP)
Isolates DNA sequences bound to proteins
- Bind proteins to DNA
- Cut DNA into short fragments
- add antibody to DNA-binding proteins for immunoprecipitation
- release proteins from DNA, re-purify DNA and sequence
Protein ChIP
Used to isolate proteins which bind to DNA sequences,
- Proteins bind to DNA
- affinity rags on DNA are used to purify DNA: protein complexes
- proteins can be eluted and sequenced
Methods of indentigying proteins bound to promoters
Edman degradation
- N-termia; sequencing
- amino acid-by-amino acid indentification
Mass spectrometry
- peptide based sequenceing
- de nono sequenceing
