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BLGY1115 > Gene Expression and Working With DNA > Flashcards

Gene Expression and Working With DNA Flashcards

1
Q

What does the promoter do?

A
  • tells the cell when / how much / in which cell to express the coding region
  • controls whether RNA polymerase engages and how many of them engage per second
  • this can be in response to e.g. nutrient availability, response to stress or disease, tissue specificity and development etc.
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2
Q

Steps in Gene Expression

A
  • gene is transcribed by RNA polymerase using the bottom strand as a template
  • the mRNA produced is identical to the top strand of the DNA, the coding strand
  • the protein is transcribed by the ribosome synthesising the polypeptide chain from the N terminus to the C terminus
  • protein folding and transport
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3
Q

Phenotype in Biochemical Terms

A

in biochemical terms the phenotype is the protein synthesised by the allele of the gene present

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4
Q

How many possible reading frames of DNA are there?

A
  • 3 possible frames if you know the orientation of the DNA

- 6 possible reading frames if you don’t know the orientation

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5
Q

Stop Codons

A
  • TAG
  • TAA
  • TGA
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6
Q

Hydrogen Bonds Between Base Pairs

A
  • 3 H bonds between C and G

- 2 H bonds between T and A

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7
Q

DNA structure

A
  • double stranded
  • antiparallel helix
  • very rigid helical structure
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8
Q

DNA vs RNA

A 
DNA
-thyamine
-H in pentose sugar
-double stranded
RNA
-uracil
-OH in pentose sugar
-single stranded
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9
Q

RNA Structure

A
  • as a single stranded molecule, RNA always wants to form double stranded structures
  • this causes folds and 3D structures to form, similar to a polypeptide chain
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10
Q

RNA Structure

Hairpin Loops

A

-if there is a section of RNA that is repeated further along the sequence but inverted then the chain will bond to itself forming a loop

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11
Q

RNA Structure

Primary Structure

A

sequence of RNA nucleotides

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12
Q

RNA Structure

Secondary Structure

A

-hairpin loops

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13
Q

RNA Structure

Tertiary Structure

A
  • more complex longer range interactions

- folding of the RNA into a compact, rigid 3D structure

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14
Q

Biological Functions of RNA Secondary Structures

A

-enable interaction with proteins
-mRNA stability and translation efficiency
-

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15
Q

Ribozyme

A

-RNA molecules acting as enzymes

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16
Q

Ribonucleoproteins

Definition

A

RNA - protein complexes

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17
Q

What are the to types of ribonucleoproteins?

A
  • ribosomes

- signal recognition particles

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18
Q

Ribosomes

A

-universal molecular machine that builds polypeptides using mRNA as a template

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19
Q

Signal Recognition Particle

A

-targets nascent proteins to either the plasma membrane (eukaryotes) or the ER membrane (prokaryotes)

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20
Q

Two Major Fates After Transcription

A

1) protein synthesis in the cytosol (SRO independent)

2) protein synthesis begins in cytosol but SRP recognises signal peptide and initiates membrane translocation

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21
Q

Gene to Protein in Prokaryotes

A
  • RNA polymerase starts transcribing
  • translation by the ribosome can begin before transcription has been completes with the ribosome following closely behind the RNA polymerase
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22
Q

Gene to Protein in Eukaryotes

A
  • transcription in nucleus produces precursor RNA
  • mRNA is then formed via distinct processing events
  • mRNA transported to cytosol
  • translated by ribosomes
  • proteins can go to many places
  • transcription termination is ill defined in eukaryotes
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23
Q

Polyadenylation Signal

A
  • only found in eukaryotic genes
  • aids in transcription termination
  • signal for certain proteins and enzymes to add the polyadenylation tail to the mRNA
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24
Q

Prokaryotic Gene Structure

A
  • promoter
  • coding region
  • transcription terminator
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25
Q

Prokaryotic Genes

Characteristice

A
  • promoter usually negatively regulated
  • usually no introns
  • one promoter can control multiple genes
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26
Q

Eukaryotic Gene Structure

A
  • promoter
  • coding region
  • polyadenylation signal
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27
Q

Eukaryotic Genes

Charcateristics

A
  • usually positively regulated
  • usually plenty of introns
  • loss of coding region subdomains
  • mono-cistronic (one promoter, one protein encoded)
  • poly a signal close to stop codon
  • does not specify transcription termination
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28
Q

Use of Alternative Sugars by E. coli

A
  • always uses glucose first
  • in the absence of glucose, E.coli takes up lactose which requires energy (just as glucose uptake does) and breaks it down to form glucose and galactose
  • to do this, genes which are not normally transcribed have to be switched on
  • the two enzymes required are, lactose permease to transport lactose into the cell, and beta galactosidase to break down the glucose
29
Q

LAC Operon

A
  • normally a repressor protein binds to the operator preventing RNA polymerase from binding to the promoter
  • the inducer, lactose, binds to the repressor protein causing a conformational change
  • the repressor protein can no longer bind to the operator so RNA polymerase can transcribe lactose permease and beta galactosidase
  • this means that there is a lag phase when the nutrient medium is changed while the required genes are induced and the enzymes are synthesised
30
Q

Passive Diffusion

A
  • follows concentration gradient

- does not require energy

31
Q

Active Transport

A
  • not spontaneous

- requires energy

32
Q

How does lactose enter the cell to perform gene induction if lactose permease hasn’t been synthesised yet?

A
  • there are some lactose permese enzymes already present in the membrane
  • as there is a constitutive basal level of transcription
  • some lactose enters via other transport proteins
33
Q

Positive Regulation

A
  • repressor protein hinders binding of RNA polymerase
  • induce stops the repressor from doing this allowing transcription to take place
  • inducer binds to the repressor
  • change in conformation of the repressor
  • repressor disengages from the DNA
  • derepressed gene
34
Q

Negative Regulation

A
  • activator protein promotes binding of RNA polymerase

- the inducer enables the activator to do this

35
Q

Attenuation

A
  • mRNA hairpin loops can terminate RNA polymerase
  • progress of the ribosome on the growing mRNA can influence this by translating the RNA before the hairpin loop can form
36
Q

LAC Operon Experiment

A
  • grow E.coli and mutagenize with UV rays or chemical mutagens
  • let lots of mutagenized cells grow with glucose as a carbon source
  • centrifuge
  • resuspend lactose medium
  • grow for less time than the lag phase
  • centrifuge
  • resuspend in glucose
  • repear until continuous growth is observed as this mean you have isolated mutants that produce lactose permease and beta galactosidase all the time so don’t require a lag phase to adjust
  • streak culture on plate producing single colonies
  • repeat growth curve with clones
37
Q

Wildtype

A

normal healthy allele

38
Q

What can mutants be used for?

A

understanding biological processes

39
Q

What are the two types of repressor mutations?

A

1) mutation that destroys the repressor (loss of function)

2) permanently active repressor, non-responsive to the inducer, this is much rarer

40
Q

Are loss of function mutations recessive or dominant?

A

recessive

41
Q

Are gain of function mutations recessive or dominant?

A

-usually dominant or partially dominant

42
Q

Constitutive Expression

Definition

A

permanent expression

43
Q

Operator Constitutive Mutation

A

operator sequence changes so that the repressor cant bind

44
Q

Defective Repressor Mutation

A

never binds even in the absence of the inducer

45
Q

Constitutively Active Repressor

A

permanently active repressor, always binds even in the presence of the inducer

46
Q

What induces the LAC operon?

A

lactose

47
Q

What represses the LAC operon?

A

glucose

48
Q

Catabolite Repression

A
  • glucose is preferred over lactose
  • in facultative autotrophic bacteria the presence of glucose or other sugars repressed genes for carbon dioxide fixation
  • this happens because the bacteria don’t have enough space for more storage of glucose so it would be a waste of energy to make more when they already have enough
49
Q

Northern Blotting

A

detecting the presence of RNA

-a very difficult process

50
Q

Western Blotting

A

detecting the presence of proteins

51
Q

Reporter Proteins

A
  • to measure gene expression, a target gene coding region can be replaced by the coding region for an enzyme that has activity that can be easily measured to work out how much transcription is occurring
  • this is a much simpler process than northern or western blotting
52
Q

Advantages of Reporter Proteins

A
  • easy detection
  • quantification is possible
  • tissue specificity
53
Q

Limitations of Reporter Proteins

A
  • moving the reporter gene into the genome can effect gene expression
  • depends on heterologous gene insertion
  • protein level regulation also takes place in cells so less of the protein may be used than is transcribed
  • reporter stability may influence may influence the result
  • is you are measuring the activity of the enzyme to determine the amount of gene expression then substrate diffusion, concentration etc. will also effect the result
54
Q

Deletion Analysis

A
  • remove sections of the promoter to find out which parts of it allow it to work
  • point mutations can also be used for more precise analysis by changing each base to see if the gene can still be transcribed
55
Q

Working With DNA vs Working With Proteins

DNA

A
  • always negatively charged
  • always hydrophilic
  • routine handling as it has constant properties
56
Q

Working With DNA vs Working With Proteins

Proteins

A
  • diverse properties, solubility, shape, charge etc.
  • mostly hydrophilic but sometimes hydrophobic
  • extraction of proteins can be difficult
  • purification is a long process and different for every protein
57
Q

Working With Plasmids

A
  • usually present in many copies
  • replicate separately from chromosomal DNA
  • easily extracted without damage
  • purified
  • modified
  • small and tough
58
Q

Plasmid

A

extrachromosomal DNA circles that replicate independently of chromosomes

59
Q

Why is it difficult to work with bacterial chromosomes?

A
  • usually attached to the membrane
  • large and break into pieces when removed
  • only one copy
60
Q

Extracting Plasmids

A
  • gentle lysis allows around half the plasmids to leave the cell without damage
  • centrifugation separated plasmids and contaminants (RNA, protein) from cell debirs, membranes and DNA
  • RNA is digested with RNase
  • proteins are removed by phenol extraction
  • DNA precipitated with salt and alcohol to clean the phenol from the DNA
  • resuspended in aqueous buffer at pH8
  • plasmids are separated on agarose gel
61
Q

Restriction Enzymes

A
  • enzymes that bacteria have to protect them from viruses
  • any restriction enzyme only ever cuts DNA at a specific sequence of bases, the restriction site
  • this cutting leaves sticky ends, exposed base pairs
62
Q

What effects the frequency of restriction sites in plasmids

A
  • frequency of restriction sites depends in plasmid size

- a typical restriction site is 6 bases long, the chance of a specific restriction site occurring is 1/4^6 = 1/4096

63
Q

Ligase Enzyme

A
  • ligase enzymes join strands of DNA together at sticky ends

- every ligase enzyme has a specific sequence of bases that it will join together

64
Q

Typical Length of Laboratory Plasmids

A

3 - 5 kbp

65
Q

Ligation

Definition

A

the joining of two strands of DNA

66
Q

Ligation of DNA from Different Plasmids

A

-plasmids are cut using restriction enzymes and centrifuges

67
Q

Vectors and Fragments

A
  • when a plasmid is cut with a restriction enzyme
  • the smaller part is called the fragment
  • the larger part is called the DNA
  • when pieces of plasmid are put back together the new plasmid that is formed is called the new recombinant plasmid
68
Q

What can go wrong with ligation of plasmids?

A
  • gene sticks back in the plasmid that it was cut from instead of the new one
  • -to prevent this use electrophoresis to separate the vector and fragment and only mix the target fragment with the target plasmid
  • polymerisation, a chain of plasmids link together forming ne long strand of DNA
69
Q

Plasmids After Ligation

A
  • once inside the bacteria, the plasmid will replicate and offer resistance to the antibiotic marker
  • bacteria are spread on plates with the appropriate antibiotic to destroy any bacteria that haven’t taken up any plasmid
  • each bacterium is allowed to grow to produce a colony of identical bacteria
  • each of these colonies is carrying different types of recombinant plasmids
  • at least one of these colonies should have the target plasmid
  • plasmids are extracted from each colony, cut and centrifuged to compare with the original plasmids
  • this allows you to identify which part of which plasmid have ended up in each colony

BLGY1115 (10 decks)

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