5 - Transcription and mRNA Vaccines Flashcards
transcription bubble
- contains RNA polymerase, DNA, and newly synthesized RNA
- where strands unwind
RNA polymerase
- make RNA from template strand
- not homologous to DNA polymerase
template strand
RNA polymerase uses this strand to make RNA complementary strand
coding strand
not read by RNA polymerase but is similar in base sequence (thymine instead of uracil) with the synthesized RNA
2 inputs of transcription
there must be two inputs in order for transcription to occur
lac operon inputs
both lactose and cAMP must be present in order for transcription to occur
promoter
region where transcription factor binds
operator
region where RNA polymerase binds
repressor in lac operon
bind to the operator, resulting in the blockage of RNA polymerase
lactose present in transcription
lactose will bind to repressor and prevent it from binding to the operator
CAP
recruits RNA polymerase and binds to operator to read DNA when in complex with cAMP
yeast 2-hybrid system
- technique to study protein-protein interactions
- activation of the reporter gene protein when there’s an interaction between bait and prey
- successful interactions will appear as a indigo dye
linkers in transcription factors
transcription proteins can be separated if linker is present, therefore cannot interact unless mediated by another interaction
bait and prey
binding of two proteins each attached to a transcription protein (BD and AD in example)
beta-galactosidase
most common reporter gene protein
X-gal
react with beta-galactosidase to produce indigo dye
transcription in eukaryotes
transcription happens in the nucleus, where the newly synthesized RNA must be processed and exported to the cytoplasm
mRNA in eukaryotes
contains 5’ cap and poly A tails added by a complex with enzyme (adenylyltransferase for polyA tails)’ post-transcriptional modifications
splicing in eukaryotes
different splicing of introns is responsible for diversity not the amount of genes
exons
expressed genes
introns
intervening sequences that must be cut out
splicing of introns
- introns have key features that will decide if it’s cut
- 2’OH in A (in branch site) will attack phosphate in the 5’ splice site creating a lariat
- OH in 5’ exon will attack phosphate in 3’ splice site, successfully cutting out the intron
cryo-electron microscopy
- gives high resolution structure of bulky, complicated molecules
- purify protein in sol’n
- dilute it in wire grid
- supercool/freeze to get particles in glassy state
- collect data with electron microscope
- analyze images as frozen particles give information on every orientation
- reconstruct 3D model (3D model will explain 2D projections taken)
mRNA vaccine
administer RNA that encodes for antigen of protein encased in a lipid particle
mRNA for COVID
- inserted RNA that encodes for spike protein found on the surface of COVID virus
- mRNA is released in cell and read by ribosome
- newly synthesized antigen will be displayed on the surface of the cell, therefore it will kill virus when encountered
Moderna’s mRNA vaccines
- chemical modification of uridine was used to increase efficacy
- chemically changing mRNA structure resulted in the change in protein expression
mRNA vaccine synthesis
- design the sequence
- put DNA that encodes with RNA polymerase, nucleotide triphosphate, etc. and perform in vitro transcription
- purify the mRNA
- encase it in lipid nanoparticles
- filter
introduction of poly A tail before transcription
DNA-encoded (encoded in DNA, therefore RNA polymerase will synthesize it)
introduction of 5’ cap before transcription
- purchase enzyme that adds 5’ cap and add it to mRNA in vitro
- provide a pre-synthesized 5’ cap which will serve as a primer, therefore RNA polymerase can just add on to it (every chemical made will have the same 5’ cap)
