Cell And Molecular (Lec 11-13)

Front 1

Are DNA binding proteins positively or negatively charged?

Back 1

Positively charged

Front 2

Where do the DNA binding proteins attach to?

Back 2

Major groove of DNA. The minor groove will give less interactions so the fit is not as tight

Front 3

Rox1 (3 points)

Back 3

-Binds to 8 sites in 3 yeast genes

-Is a transcription factor

-Sites aren't identical - different sites have different affinity for the protein

Front 4

Helix-turn-helix DNA binding motif (2 points

Back 4

-Recognition helix inserts into major groove - specific contact with DNA

-Bind as dimers

Front 5

Zinc finger DNA binding motif (4 points)

Back 5

-4 amino acids hold the zinc atom

-Alpha helix recognises 2 bases

-Major groove contact

-Strong DNA-protein interaction

Front 6

Leucine zipper binding motif (2 points)

Back 6

-Monomer held by leucine - hydrophobic

-Can form homo- or heterodimers

Front 7

Helix-loop-Helix

Back 7

-Has a flexible loop with no structure but similar to Leucine zipper

-Attaches to DNA recognition domain

-Increases binding strength

Front 8

DNAse1 footprinting (identifying DNA binding proteins (3 points)

Back 8

-Label 1 end of DNA and mix with cell extract

-Add DNAse to partially digest DNA

-Heat to destroy DNAse and release binding proteins

= identify where a protein binds on DNA

Front 9

Electrophoretic mobility shift assay (EMSA) (identifying DNA binding proteins (3 points)

Back 9

-Label DNA and run on its own

-Mix with cell extract and run on gel electrophoresis

-Unbound DNA migrates slower in the DNA

= study interactions and what is involved in binding complex

Front 10

Transcription factors can be...

Back 10

Permissive

Specific

Regulatory

Front 11

There are two types of regulatory transcription factors

Back 11

Activators - increase transcription of genes

Repressors - decrease transcription of genes

Front 12

DNA looping

Back 12

Chromatin does not bed easily and allows transcription around the gene

Front 13

Insulator (2 points)

Back 13

blocks interaction between enhancers and promoters

block regulatory sequences from affecting neighbouring genes

Front 14

What can alter gene expression? (4 points)

Back 14

-Weakly activating protein assembly

-Strongly activating assembly

-Strongly inhibiting protein

-Spacer DNA

Front 15

Genetic switch

Back 15

The genes are on and a conformational change occurs that turns them off

Front 16

How can you regulate a transcription factor? (6 points)

Back 16

-Protein synthesis

-Protein phosphorylation

-Unmasking

-Ligand binding

-Addition of second subunit

-Stimulation of nuclear entry

Front 17

How do transcription factors interact?

Back 17

Synergistically

Front 18

Genetic alterations to the DNA sequence can permanently affect gene expression

Back 18

Epigenetic changes can modulate gene expression but do not alter DNA sequence and are reversible

Front 19

Nucleosomes can be covalently modified - what will this do?

Back 19

This will affect gene transcription

Front 20

Competing modifications in histone tails

Back 20

Histone acetyl transferases (HATS) - modify lysine residues

Histone methyl transferases (HMTS) - exhibit site-specifications

these are reversible

Front 21

What do histone modifications signify?

Back 21

Distinct states of transcriptional activity or competence

Front 22

Acetylation of histones

Back 22

-Create binding sites for transcriptional activation factors which contain a bromodomain

Front 23

Methylation

Back 23

-Create binding sites for transcriptional repressors with a chromodomain

-Also activators with a zinc finger domain

Front 24

Role of transcription activator proteins in chromatin

Back 24

-Selective nucleosome remodelling

-Histone removal

-Promotes RNA polymerase recruitment

Front 25

Role of transcription repressor proteins in chromatin

Back 25

-Competitive DNA binding

-Recruitment of chromatin remodelling complexes

Front 26

Polycomb group of proteins - their role and examples

Back 26

-These generate repressive chromatin modifcations

-PCR1 - recognises mark = maintains repressed state

-PCR2 - makes the mark = triggers transcriptional repression

Front 27

What is the addition of methyl groups to cytosine residues mediated by?

Back 27

DNA methyltransferases (DNMTs)

Front 28

Mammalian X-chromosome inactivation - how it occurs (4 points)

Back 28

The levels of X-chromosome derived gene products

-Males - XY - 1 dose of x-linked genes

-Females - XX - 2 doses of x-linked genes

-Only X chromosome is silence

-Initial selection for silencing is random

-Some will inherit the same silenced X chromosome

Front 29

X chromosome inactivation example (4 points)

Back 29

Calico cats

-always be female

-Heterozygous for 2 alleles of X-linked coat pigment gene

-Patches are due to random X-inactivation

-involves synthesis of non-coding RNA

Front 30

Barr body

Back 30

Supercoiled inactive X chromosome at periphery of nucleus of female somatic cells - occurs in calico cats

Front 31

The genetic code

Back 31

-non-overlapping

-degenerate

-fixed starting point = AUG = methionne

-tRNA is template for amino acids - caries an anti-codon loop

Front 32

Wobble bases

Back 32

Allow same anticodon to bind to more than one codon

Front 33

What is coupling of amino acid to tRNA achieved by?

Back 33

Aminoacyl-tRNA synthetase

Front 34

Coupling to the correct amino acid/tRNA

Back 34

-requires two adaptors

-Synthetase is required

-pairing needs highly-specific interactions

Front 35

Where are new amino acids added during protein synthesis? Where does this occur?

Back 35

C-terminus

Occurs in the ribosome

Front 36

Elongation factors

EF-1 (3 points)

Back 36

Help translation and improve accuracy

-Binds to tRNA

-Causes two delays before peptidyl transferase can act (hydrolysis of GTP and dissociation from tRNA)

Front 37

What happens if there is an absence of EF-1?

Back 37

There are more errors in protein sequence

Front 38

What helps met tRNA bind to small ribosome subunits? How does this occur?

Back 38

ELF-2

-Complex binds to cap and associated initiation factors

-Scans along mRNA until AUG is found

-ELF-2 is released and ribsome forms

Front 39

Folding of protein (2 points)

What happens if there is an incorrect step?

Back 39

Puts hydrophobic side chains in the middle to achieve a lower energy state

If there is the correct conformation this leads to a molten globule

It may reduce the energy state and block further folding

Front 40

What is the role of molecular chaperones? (2 points)

Back 40

They reverse incorrect steps

Their expression is elevated when the temperature is above normal

Front 41

HSP70 (two points)

Back 41

-works directly on proteins as they exit the ribosome

-bind to exposed hydrophobic amino acids

Front 42

HSP60 (two points)

Back 42

-puts misfolded proteins into isolation

-a cap seals protein and allows refolding

Front 43

Diseases can be caused by protein aggregates - what are examples?

Back 43

Huntington's and Alzheimer's Disease

Front 44

Amyloid plaques

Back 44

made of cross-beta filaments

Front 45

Creutzfeldt-Jakob disease (CJD)

Back 45

-Proin is present and joins with amyloid to create heterodimer = forms cross-beta filaments

-Creates holes in and around neurons in the brain