Lecture 13 Flashcards
eukaryotic gene regulation
splicing, transcription, etc
-can also degrade mRNA reducing its lifespan
biochemical assay
measure mRNA
-have to create DNA template that has a promotoer and coding region
-open the cells you are using and add radiolabelled NTP and proteins
-look for radiolabeled RNA
what cells are used for biochemical assay
dead cells
northern blot
mRNA
-separates based on size
western blot
protein
what is for live cells?
creating a transgenic organism with the genes that we want
Steps of transgenic
-replace the coding region with a reporter gene so when the promoter is active the reporter is active
-popular is lac Z
-a color change can indicate that the promoter is active (GFP maybe)
reverse transcriptase comes from?
retrovirus that uses it to make DNA from their mRNA once in the host
RT-PCR
-uses RNA as a template
-makes the complementary DNA
-only makes one strand so we need DNA pol to make second
-then PCR to amplify DNA
what is less stable DNA or RNA
RNA
RT-QPCR
-quantitative measure of mRNA
cDNA microarray
measures all/ many genes at once
-uses RT with flourecent DNTP
-can make experimental and control groups different colors
-then onto microarray chip
RNA sequence
-convert to cDNA and sequence the whole thing
-the number of reads will be proportional to the DNA you started with
initiation
-RNA pol goes to the promoter
-transcription factors bind to core promoter and attract RNA pol
does RNA pol need 5 primer
no it just needs dsDNA
where is promoter
upstream to start point
-tells the start point
what happens when RNA pol binds
transcription initiation complex
-BUT transcription has not started yet
core promoter
TATA BOX
-10 bases upstream
-in bacteria
eukaryote TATA
-25 bases upstream
-not the only thing in that region
where does transcription start
at 0 base where there is an UTR
is the UTR turned to protein
no
-it is the site for ribosome binding for translation
where is shine dalgaro
+30
what makes a promoter strong?
having a resemblense to the sequence/ recruiting RNA pol bc this promoter would not need CAP
-regulates transcription
what determines promoter specificity
sigma subunit
sigma 70
most common
-recognizes -30 to -10
-binds to promoter
sigma 32
heat response protein
-becomes part of RNA pol activating gene to let bacteria survive heat
sigma 54
nitrogen starvation
-different promoter
one sigma factor…
encodes multiple genes
elongation
RNA unwinds helix in a bubble, slower than RNA pol, no helicase
T or F only one RNA pol can be active at a time
-false you can have many they just cant be on the promoter region together
sense strand
leading strand, for mRNA
how does RNA pol find mistakes?
it doesn’t
-RNA pol cant proofread like DNA pol
-the error is not int he genome like with DNA pol so less serious
-redundancy of code might even make the mistake insignificant
-splicing can also remove mistake
