PCR, Transcription, Translation Flashcards
1
Q
What polymerase is used in PCR?
A
Taq polymerase
2
Q
What requirements are in both PCR and DNA Replication
A
Parent/ template strand
Polymerase
dNTPs and mg2+
primer with free 3’)H
3
Q
What is the PCR Master Mix?
A
- contains all PCR requirements except DNA
- faster, less chance of error and contamination
- primers, dNTPs, mg2+, polymerase, buffer, water
4
Q
What are the 3 steps of PCR?
A
- Denaturation, dsDNA -> ssDNA
- Annealing, primers anneal to ssDNA
- Elongation, Taq Pol elongates the daughter strand
5
Q
Denaturation:
- what occurs in this step?
- Why do we do this step?
- What temp do we do this step?
- What does the temp depend on?
A
- break down double helix into single strands
- hydrogen bonds between base pairs break
- 94 degrees
- temp depends on GC content
6
Q
How is Taq DNA thermostable?
What is another example of a thermostable DNA polymerase?
A
- can resist high temps
- first isolated from a thermostable bacterium
- Pfu Pol
7
Q
Annealing:
- What/ why occurs in this step?
- What temp is required?
- What is the duration of this step?
A
- primers anneal to ssDNA
- 55 degrees
- lower temp allows this
- 30s to 1min
8
Q
- What are PCR Primers?
- How long are they (bp)?
- What is the forward and reverse primer?
- Why can they be incorporated into the sequence?
A
- short ssDNA, complementary to flanking region
- 15 - 30bps
- anneal 5’-3’ on opposite strands, extend in opposite directions
- don’t contain RNA
9
Q
Extension/ Elongation:
- What happens in this step?
- What co-factor is required?
- What is the ideal temperature?
- What is its duration? How can this vary?
A
- Taq pol elongates daughter strands
- mg2+ needed
- 72 degrees
- depends on the length of the target sequence, Taq does 1kb per min
10
Q
How do we calculate the exponential amplification of DNA in PCR?
A
2^n
n = number of cycles
11
Q
Why do we have an initial duration and additional elongation step?
A
- 5 min at 92 degrees, ensures denaturation
- 10 mins at 72 degrees, ensures full extension and reduced truncated PCR products
12
Q
How are PCR products analysed on an agarose gel?
A
- knowing the expected size of the target sequence
- DNA ladder
13
Q
What is a PCR negative control?
A
- a test to check for DNA contamination in PCR reagents
- run on gel, no DNA bands should show up
14
Q
Would PCR work on an RNA template?
What is an alternative technique?
A
- no. DNA pol doesn’t read RNA and would need NTPs not dNTPs
- reverse transcriptase could be used in PCR, would result in cDNA, a DNA version of the RNA
15
Q
What enzyme carries the transcription of DNA to RNA?
A
RNA polymerase
16
Q
Draw a venn diagragm compreing DNA and RNA. polymerases.
- 5 similarities
- 2 difference
A
- uses nucleoside 5’ triphos precursors
- catalyses phosphodiester bond
- uses DNA as templates
- base pairing required
- grows 5’-3’
- uses RIBOnucleosie precursors
- no primer needed
17
Q
Discuss 2 features of bacterial RNA polymerase
A
- core tetramer
- sigma subunit initiates transcription
18
Q
What are the 4 main steps of transcription?
A
- binding at OriC
- initiation
- elongation
- termination at Ter site
19
Q
How is transcription started? / Compare the sense and antisense strand
A
Starts at initiation point on non-coding/ antisense/ temp DNA strand. The other strand, that has same sequence ar RNA = coding/ sense DNA strand
20
Q
What are operons? Do we have then in eukaryotic organisms? Introduce the lactose operon.
A
- way of grouping genes
- when transcribed will induce a similar process
- lactose operons, Z, Y, A
21
Q
Discuss step 1 of transcription: RNA Pol binding
- how are promoters recognised
- how long are these sites (e.coli)
- where are they located
- what is the “upstream” and “downstream” end
- what is a common sequence
A
- recognised by sigma factor
- 40bp
- on coding 5’ strand
- up = sequence before start of transcription, down = +1 after
- -35 and -10
22
Q
Discuss step 2 of transcription: promoter recognition and transcription initiation
A
RNA Pol binds loosely to DNA, quickly runs down until sigma recognises corresponding promoter, binds tightly, sigma dissociates, elongates 5’-3’
23
Q
Discuss step 3 of transcription: Elongation
A
- DNA unwinds ahead and rewinds behind RNA Pol, creates 17bp “transcription bubble”
24
Q
How is transcription terminated in bacteria?
A
- term signal (using eg of e.coli)
- right C-G, followed by 4 As
- weak H-bonding releases RNA “hair-pin” from DNA and enzyme
25
What important consideration allows transcription and translation to happen faster in bacteria?
No nucleus or cellular boundaries
26
What 2 key differences between transcription in bacteria and eukaryotes?
Eukaryotes have multiple RNA Pols and more complex control sequences
27
Describe Eukaryotic RNA Pols;
- RNA Pol l
- RNA Pol ll
- RNA Pol lll
- others
1. nucleoli, make rRNA precursors
2. nucleoplasm, makes mRNA precursors
3. nucleoplasm, makes 5s rRNA precursors, tRNA, and other
* Also has separate mitochondrial and chloroplast pol
28
What do eukaryotes use instead of the sigma factor to recognise different promoters?
- accessory proteins
- e.g. transcription factors
- more complex and diverse than sigma
29
Discuss RNA Pol ll (Eukaryotic);
- constitutively v selectively transcribed
- TATA box
- CCAAT box
- Constitutively transcribed genes are "house-keeping genes" that are always on and expressed in all cell types
- Selectively transcribed genes are expressed in fewer cell types are are turned on and off
- TATA box is a core element of the promotor region, it is similar to the -10 promotor sequence in bacteria and is approx 25-25bp upstream.
- CCAAT box regulates the transcription rate and it is located -70 and -90
30
What are 3 important sequence elements in a eukaryotic gene?
- TATA box: binds TBP
- CAAT: binds CTF
- GC: binds Sp1
31
Discuss other regulatory elements: What, who where?
What: Determine frequency and efficiency, accessory proteins can bind and then activate or repress
Who: Enhancers, SIlencers, response elements (targets sequences)
Where: up or downstream
32
What is post-transcription RNA processing why is it needed?
- no precise Ter sites as in bacterial
- allows for post-modification process to acquire biological activity
- mRNA, tRNA, rRNA
33
What happens in mRNA processing?
- 5' capping
- 3' polyadenylation
- splicing
Capping:
- protects from 5' exonuclease
- several enzymatic processes
- adds 7-MethylG group
- added when approx 30 bp long
Polyadenylation:
- protects from 3' exonuclease
- ATP action of Poly-A-polymerase from floating As (approx 250)
- Handle for proteins to carry to the ribosome
- spliced introns, non-coding sequences
Splicing:
- not in bacterial processing
- coding and non-coding genes are interspersed
- introns are excised, exons splice
34
Discuss splicing
- enzyme used
- alternative
- spliceosome (5x small nuclear riboproteins and min100 associated proteins)
- can recognise conserved sequences at exon/intron junctions
- tiff tissues splice differently
- one sequence can yield many diff. proteins
35
What is meant by the genetic code?
correspondence between nucleic acid and polypeptide sequences
36
What are codons?
- how many bases per?
- how many different triplets?
- what are reading frames? how are they properly read?
- 3 bases per
- 64 triplet sequences
- reading frames are a way of sequentially dividing mRNA into consecutive triplets, codons, which will translate into amino acids
- selection of the proper reading frame depends on the precise identification of a translation start site
37
Why is the genetic code degenerate and non-random? Give an example using start and stop codons.
- degenerate code is when several code words have the same meaning, in the same way, many different codons can signal a particular amino acid
- non-random as the third codon position doesn't have a huge impact.
- start: AUG, GUG
- stop: UAG, UAA, UGA
38
Discuss tRNAs;
- purpose
- length
- structure (acceptor stem and anticodon loop)
- carry aa and recognise the corresponding codon
- 54-100 nucleotides
- secondary cloverleaf
- or tertiary L-shape
- acceptor stem (aa)
- anticodon loop (reads mRNA codon)
- has post-transcriptionally modified bases; helps attachment of aa and codon interaction
39
What occurs in tRNA Aminoacylation? (2 steps + enzyme involved)
- aaRS appends aa to tRNA
1. aa + ATP reaction activated aa into aminoacyl-AMP
2. aminoacyl-AMP + tRNA = aa-tRNA
40
How does an aaRS recognise a tRNA so that it can be charged with the correct amino acid?
- aaRS can recognise unique structural features
- proof-reading mechanism aids fidelity
41
What are 6 functions of the ribosome?
1. site of protein synthesis
2. binds mRNA so codons can be read
3. has tRNA binding sites
4. mediates the interaction of non-ribosomal protein factors
5. enzyme action, catalyses formation of peptide bonds
6. can move to translate
42
Give an overview of Ribosome structure
- 30s small subunit, 50s large subunit
- A, P, E-sites
43
Compare the small and large subunit of the bacterial ribosome?
30s - small
- 16 rRNA
- 21 proteins
50s - large
- 5s and 23s rRNA
- 31 proteins
44
Discuss the 3 main sites on ribosomes that tRNA bind to.
A-site: aa-tRNA
P-site: peptidyl tRNA
E-site: deacylated tRNA
45
Compare the eukaryotic ribosome and bacterial ribosome?
bacterial = 70s
eukaryotic = 80s
46
Discuss the small and large subunits in rat liver cytoplasmic ribosome.
40s small
60s large
47
Translation overview;
- How does polypeptide synthesis proceed?
- How does the chain occur?/ How are polypeptides added?
- Which direction is mRNA read?
- How does translation occur in polysomes?
- N to C terminus
- chain is added to new aa
- 5' --> 3'
- on polysome, ribosome binds to mRNA --> polyribosome
48
How are peptide bonds formed with reference to the ribosomal peptidyl transferase reaction?
peptidyl-tRNA in P-site, added onto aa-tRNA in A-site
49
In bacterial systems, how does it know that AUG codes a start and not a regular Met aa?
- Shine-Dalgarno sequence
- approx 10 baes upstream
- ribosomal binding site
** Not in Eukaryotic
50
Discuss chain initiation in bacteria.
- 2 ribosomal subunits assemble with fMet-tRNA on mRNA
- requires initiation factors (IF-1, IF-2, IF-3, in E.coli)
51
Discuss chain elongation in bacteria + draw a diagram;
1. Decoding
2. Transpeptidation
3. Translocation
- the rate that this occurs
1. (GTP) tRNA binds to matching codon (A-site)
2. Peptide bond formation. The polypeptide (P-site) is linked to a new amino acid (A-site)
3. (GTP) mRNA shifts forward, new codon exposed
10-20 amino acid residues per second.
52
What are the 3 elongation factors?
Tu
Ts
G
53
What is post-translational processing?
- Folding of protein. Assisted by chaperones
1. Proteolysis (braking peptide bonds)
2. Covalent modification (enzyme-catalysed alterations)
3. Translocation (move through a membrane)
4. Glycosylation (dictate proper confirmation)
54
How are antibiotics useful in undersanding ribosomal mechanisms?
- most block translation
- by blocking a process, allows more detailed analysis
55
Discuss streptomycin.
- drug class
- low conc. consequences
- high conc. consequences
- aminoglycoside
- Low: causes ribosomes to misread mRNA and inhibit the growth of susceptible cells (but not kill them)
- High: prevents chain initiation, causing cell death
56
Discuss chloramphenicol.
- drug class
- cell activity
- clinical use
- broadspectrum
- inhibits peptidyl transferase (formation of peptide bonds)
- binds to large subunit near A-site
- used in conjunctivitis, although overall, limited clinical use due to side effects
57
Discuss Rifampicin.
- inhibit bacterial DNA-dependent RNA polymerase
- drug binding in the polymerase subunit deep within the DNA/RNA channel, facilitating direct blocking of the elongating RNA
