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.
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
3
Q
Phenotype in Biochemical Terms
A
in biochemical terms the phenotype is the protein synthesised by the allele of the gene present
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
5
Q
Stop Codons
A
- TAG
- TAA
- TGA
6
Q
Hydrogen Bonds Between Base Pairs
A
- 3 H bonds between C and G
- 2 H bonds between T and A
7
Q
DNA structure
A
- double stranded
- antiparallel helix
- very rigid helical structure
8
Q
DNA vs RNA
A
DNA -thyamine -H in pentose sugar -double stranded RNA -uracil -OH in pentose sugar -single stranded
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
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
11
Q
RNA Structure
Primary Structure
A
sequence of RNA nucleotides
12
Q
RNA Structure
Secondary Structure
A
-hairpin loops
13
Q
RNA Structure
Tertiary Structure
A
- more complex longer range interactions
- folding of the RNA into a compact, rigid 3D structure
14
Q
Biological Functions of RNA Secondary Structures
A
-enable interaction with proteins
-mRNA stability and translation efficiency
-
15
Q
Ribozyme
A
-RNA molecules acting as enzymes
16
Q
Ribonucleoproteins
Definition
A
RNA - protein complexes
17
Q
What are the to types of ribonucleoproteins?
A
- ribosomes
- signal recognition particles
18
Q
Ribosomes
A
-universal molecular machine that builds polypeptides using mRNA as a template
19
Q
Signal Recognition Particle
A
-targets nascent proteins to either the plasma membrane (eukaryotes) or the ER membrane (prokaryotes)
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
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
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
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
24
Q
Prokaryotic Gene Structure
A
- promoter
- coding region
- transcription terminator
25
Prokaryotic Genes
| Characteristice
- promoter usually negatively regulated
- usually no introns
- one promoter can control multiple genes
26
Eukaryotic Gene Structure
- promoter
- coding region
- polyadenylation signal
27
Eukaryotic Genes
| Charcateristics
- 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
28
Use of Alternative Sugars by E. coli
- 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
LAC Operon
- 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
Passive Diffusion
- follows concentration gradient
| - does not require energy
31
Active Transport
- not spontaneous
| - requires energy
32
How does lactose enter the cell to perform gene induction if lactose permease hasn't been synthesised yet?
- 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
Positive Regulation
- 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
Negative Regulation
- activator protein promotes binding of RNA polymerase
| - the inducer enables the activator to do this
35
Attenuation
- 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
LAC Operon Experiment
- 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
Wildtype
normal healthy allele
38
What can mutants be used for?
understanding biological processes
39
What are the two types of repressor mutations?
1) mutation that destroys the repressor (loss of function)
| 2) permanently active repressor, non-responsive to the inducer, this is much rarer
40
Are loss of function mutations recessive or dominant?
recessive
41
Are gain of function mutations recessive or dominant?
-usually dominant or partially dominant
42
Constitutive Expression
| Definition
permanent expression
43
Operator Constitutive Mutation
operator sequence changes so that the repressor cant bind
44
Defective Repressor Mutation
never binds even in the absence of the inducer
45
Constitutively Active Repressor
permanently active repressor, always binds even in the presence of the inducer
46
What induces the LAC operon?
lactose
47
What represses the LAC operon?
glucose
48
Catabolite Repression
- 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
Northern Blotting
detecting the presence of RNA
| -a very difficult process
50
Western Blotting
detecting the presence of proteins
51
Reporter Proteins
- 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
Advantages of Reporter Proteins
- easy detection
- quantification is possible
- tissue specificity
53
Limitations of Reporter Proteins
- 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
Deletion Analysis
- 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
Working With DNA vs Working With Proteins
| DNA
- always negatively charged
- always hydrophilic
- routine handling as it has constant properties
56
Working With DNA vs Working With Proteins
| Proteins
- 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
Working With Plasmids
- usually present in many copies
- replicate separately from chromosomal DNA
- easily extracted without damage
- purified
- modified
- small and tough
58
Plasmid
extrachromosomal DNA circles that replicate independently of chromosomes
59
Why is it difficult to work with bacterial chromosomes?
- usually attached to the membrane
- large and break into pieces when removed
- only one copy
60
Extracting Plasmids
- 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
Restriction Enzymes
- 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
What effects the frequency of restriction sites in plasmids
- 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
Ligase Enzyme
- 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
Typical Length of Laboratory Plasmids
3 - 5 kbp
65
Ligation
| Definition
the joining of two strands of DNA
66
Ligation of DNA from Different Plasmids
-plasmids are cut using restriction enzymes and centrifuges
67
Vectors and Fragments
- 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
What can go wrong with ligation of plasmids?
- 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
Plasmids After Ligation
- 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
