Lecture 13

Question 1

eukaryotic gene regulation

Answer 1

splicing, transcription, etc
-can also degrade mRNA reducing its lifespan

Question 2

biochemical assay

Answer 2

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

Question 3

what cells are used for biochemical assay

Answer 3

dead cells

Question 4

northern blot

Answer 4

mRNA
-separates based on size

Question 5

western blot

Answer 5

protein

Question 6

what is for live cells?

Answer 6

creating a transgenic organism with the genes that we want

Question 7

Steps of transgenic

Answer 7

-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)

Question 8

reverse transcriptase comes from?

Answer 8

retrovirus that uses it to make DNA from their mRNA once in the host

Question 9

RT-PCR

Answer 9

-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

Question 10

what is less stable DNA or RNA

Answer 10

RNA

Question 11

RT-QPCR

Answer 11

-quantitative measure of mRNA

Question 12

cDNA microarray

Answer 12

measures all/ many genes at once
-uses RT with flourecent DNTP
-can make experimental and control groups different colors
-then onto microarray chip

Question 13

RNA sequence

Answer 13

-convert to cDNA and sequence the whole thing
-the number of reads will be proportional to the DNA you started with

Question 14

initiation

Answer 14

-RNA pol goes to the promoter
-transcription factors bind to core promoter and attract RNA pol

Question 15

does RNA pol need 5 primer

Answer 15

no it just needs dsDNA

Question 16

where is promoter

Answer 16

upstream to start point
-tells the start point

Question 17

what happens when RNA pol binds

Answer 17

transcription initiation complex
-BUT transcription has not started yet

Question 18

core promoter

Answer 18

TATA BOX
-10 bases upstream
-in bacteria

Question 19

eukaryote TATA

Answer 19

-25 bases upstream
-not the only thing in that region

Question 20

where does transcription start

Answer 20

at 0 base where there is an UTR

Question 21

is the UTR turned to protein

Answer 21

no
-it is the site for ribosome binding for translation

Question 22

where is shine dalgaro

Answer 22

+30

Question 23

what makes a promoter strong?

Answer 23

having a resemblense to the sequence/ recruiting RNA pol bc this promoter would not need CAP
-regulates transcription

Question 24

what determines promoter specificity

Answer 24

sigma subunit

Question 25

sigma 70

Answer 25

most common
-recognizes -30 to -10
-binds to promoter

Question 26

sigma 32

Answer 26

heat response protein
-becomes part of RNA pol activating gene to let bacteria survive heat

Question 27

sigma 54

Answer 27

nitrogen starvation
-different promoter

Question 28

one sigma factor…

Answer 28

encodes multiple genes

Question 29

elongation

Answer 29

RNA unwinds helix in a bubble, slower than RNA pol, no helicase

Question 30

T or F only one RNA pol can be active at a time

Answer 30

-false you can have many they just cant be on the promoter region together

Question 31

sense strand

Answer 31

leading strand, for mRNA

Question 32

how does RNA pol find mistakes?

Answer 32

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