Lecture 10 Slides Flashcards Preview

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Flashcards in Lecture 10 Slides Deck (36):
1

Lysosomes and vacuoles

Digestive organelles
Lysosomes digest proteins in organelles
Vacuoles we associate with plants

2

How do I know if my gene is expressed? Where can I test for its expression?

Stages are
DNA replication
transcription
MRNA
Translation
protein

You can test post transcription or post translation

3

Suppose you are looking for a specific protein? What test can you use to see if you've found it?

Western blotting

4

Why can't you use PCR with mRNA?

It is not a template for DNA

5

What can you use to test for mRNA?

Microarray
Northern blotting
RTPCR?

6

PCR

Polymerase chain reaction
Amplification
DNA is copied
Denaturion 95 degrees
Annealing 55-65 degrees
Extension 72degrees
Repeat

7

RTPCR

Reverse transcriptase PCR
Detects RNA expression
Create cDNA, has no introns
Use RNA and enzyme to create cDNA, on which you can use PCR

8

Glyceraldehydethreephosphate (GAPDH)

Housekeeping gene used as reference, control

9

What is most important regulatory step

Transcription controls
BY DNA regulatory proteins

10

Which subunits do DNA subunits usually have

1. Binds DNA
2. Activator/repressor domain

11

How does protein subunit bind dna

Usually via h bonding to bases, ionic bonds with backbone

12

How do protein subunits activate or suppress

Bind RNA polymerase II or other proteins (how modular transcription)

13

How does binding happen between protein subunits

Alpha helices with major groups in DNA

14

Why's codon is at start site

ATG

15

Upstream regulatory element(URE)

A short sequence of 6-12 bps "cis"

16

Homeodomain

Part of protein that binds to specific regulatory regions of target genes.

17

Homeobox

DNA sequence found within genes that are involved in the regulatory patterns of anatomical development in animals, fungi, and plants

18

Benefit of having regulatory sequences and multiple conning sequences on genome

Reduces number of monomers you have to make

19

How do I know what DNA my transcription factor binds to?

Chromatin immunoprecipitation(CHIP seq)
DNA sequencing

Primary tissue treated with formaldehyde to covalently cross link DNA and proteins

Protein specific antibodies are added to immunoprecipitates to target protein
Proteins are then unlinked and DNA is purified


DNA fragments are amplified and fluorescently tagged


A library is constructed


Fragments are sequenced

Sequences are aligned to a reference of genome and then can be visualized

20

How many proteins does a promoter bind to

Hundreds

21

How is regulation of prokaryotic transcription simple

Most controls are at or near promoter
Just one polymerase bacterium

22

What is a repressor? How does it work?

A protein that blocks RNAP from binding to promoter

23

What does repressor usually bind to

Operator sequence

24

What is activator? How does it work?

A protein that helps recruit RNAP to active site

25

How is regulation of eukaryotic dna complex

Eukaryotic DNA is flexible
UREs can be 50+ kb from +1
Many proteins are involved and interact together

26

TATA box

DNA sequence that indicates where a genetic sequence can be read and decoded. Type of promoter sequence. Says where transcription begins.

27

Example of how many proteins are involved in eukaryotic transcription

RnaII binds to TATA. A mediator binds around RNAII. If DNA is flexible and bent, two activators at UREs far away can recruit RNAII/mediator to promoter. Somewhere closer, URE can have a repressor on it so as not to express its sequence.

28

Three abilities of eukaryotic repressors

Can compete for activator binding site
Can inhibit activator interaction with RNAPII
inhibit RNAPII directly as well

29

Three ways to get rid of bound repressors

Conformational change
Phosphorylation
Ubi ligase (plants and animals)

30

Epi genetics

Heritable changes in gene expression that does not involve changes to the underlying DNA sequence; a change to phenotype without a change to genotype.

31

How can epigenetic mechanisms work

In cis -affect only one chromosomal copy
In trans - two self propagating mechanisms that work, affecting both chromosomal copies of a gene.

32

Epigenetic mechanisms that act in cis

DNA methylation
Histone modification

33

Epigenetic mechanisms that act in trans

Positive feedback loop by transcription regulator
Protein aggregation state

34

What do transcription activators recruit? 4

Histone acetyl transferase (HATs)
Histone kinases
Histone chaperones
Chromatin remodeling complexes

35

What do transcription repressors recruit?

Histone deacetylase
Histone methyl transferase
Chromatin remodeling complexes to create more heterochromatin and prevent gene expression

36

Proteozomes

Cytosolic proteins