5 - Transcription and mRNA Vaccines

Question 1

transcription bubble

Answer 1

• contains RNA polymerase, DNA, and newly synthesized RNA

- where strands unwind

Question 2

RNA polymerase

Answer 2

• make RNA from template strand

- not homologous to DNA polymerase

Question 3

template strand

Answer 3

RNA polymerase uses this strand to make RNA complementary strand

Question 4

coding strand

Answer 4

not read by RNA polymerase but is similar in base sequence (thymine instead of uracil) with the synthesized RNA

Question 5

2 inputs of transcription

Answer 5

there must be two inputs in order for transcription to occur

Question 6

lac operon inputs

Answer 6

both lactose and cAMP must be present in order for transcription to occur

Question 7

promoter

Answer 7

region where transcription factor binds

Question 8

operator

Answer 8

region where RNA polymerase binds

Question 9

repressor in lac operon

Answer 9

bind to the operator, resulting in the blockage of RNA polymerase

Question 10

lactose present in transcription

Answer 10

lactose will bind to repressor and prevent it from binding to the operator

Question 11

CAP

Answer 11

recruits RNA polymerase and binds to operator to read DNA when in complex with cAMP

Question 12

yeast 2-hybrid system

Answer 12

• 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

Question 13

linkers in transcription factors

Answer 13

transcription proteins can be separated if linker is present, therefore cannot interact unless mediated by another interaction

Question 14

bait and prey

Answer 14

binding of two proteins each attached to a transcription protein (BD and AD in example)

Question 15

beta-galactosidase

Answer 15

most common reporter gene protein

Question 16

X-gal

Answer 16

react with beta-galactosidase to produce indigo dye

Question 17

transcription in eukaryotes

Answer 17

transcription happens in the nucleus, where the newly synthesized RNA must be processed and exported to the cytoplasm

Question 18

mRNA in eukaryotes

Answer 18

contains 5’ cap and poly A tails added by a complex with enzyme (adenylyltransferase for polyA tails)’ post-transcriptional modifications

Question 19

splicing in eukaryotes

Answer 19

different splicing of introns is responsible for diversity not the amount of genes

Question 20

exons

Answer 20

expressed genes

Question 21

introns

Answer 21

intervening sequences that must be cut out

Question 22

splicing of introns

Answer 22

• 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

Question 23

cryo-electron microscopy

Answer 23

• gives high resolution structure of bulky, complicated molecules

1. purify protein in sol’n
2. dilute it in wire grid
3. supercool/freeze to get particles in glassy state
4. collect data with electron microscope
5. analyze images as frozen particles give information on every orientation
6. reconstruct 3D model (3D model will explain 2D projections taken)

Question 24

mRNA vaccine

Answer 24

administer RNA that encodes for antigen of protein encased in a lipid particle

Question 25

mRNA for COVID

Answer 25

• 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

Question 26

Moderna’s mRNA vaccines

Answer 26

• chemical modification of uridine was used to increase efficacy
• chemically changing mRNA structure resulted in the change in protein expression

Question 27

mRNA vaccine synthesis

Answer 27

1. design the sequence
2. put DNA that encodes with RNA polymerase, nucleotide triphosphate, etc. and perform in vitro transcription
3. purify the mRNA
4. encase it in lipid nanoparticles
5. filter

Question 28

introduction of poly A tail before transcription

Answer 28

DNA-encoded (encoded in DNA, therefore RNA polymerase will synthesize it)

Question 29

introduction of 5’ cap before transcription

Answer 29

• 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)