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Bioc2201 > PCR, Transcription, Translation > Flashcards

PCR, Transcription, Translation Flashcards

1
Q

What polymerase is used in PCR?

A

Taq polymerase

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2
Q

What requirements are in both PCR and DNA Replication

A

Parent/ template strand
Polymerase
dNTPs and mg2+
primer with free 3’)H

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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
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4
Q

What are the 3 steps of PCR?

A
  1. Denaturation, dsDNA -> ssDNA
  2. Annealing, primers anneal to ssDNA
  3. Elongation, Taq Pol elongates the daughter strand
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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
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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
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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
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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
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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
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10
Q

How do we calculate the exponential amplification of DNA in PCR?

A

2^n
n = number of cycles

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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
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12
Q

How are PCR products analysed on an agarose gel?

A
  • knowing the expected size of the target sequence
  • DNA ladder
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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
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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
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15
Q

What enzyme carries the transcription of DNA to RNA?

A

RNA polymerase

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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
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17
Q

Discuss 2 features of bacterial RNA polymerase

A
  • core tetramer
  • sigma subunit initiates transcription
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18
Q

What are the 4 main steps of transcription?

A
  1. binding at OriC
  2. initiation
  3. elongation
  4. termination at Ter site
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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

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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
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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
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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’

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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
Q

What important consideration allows transcription and translation to happen faster in bacteria?

A

No nucleus or cellular boundaries

26
Q

What 2 key differences between transcription in bacteria and eukaryotes?

A

Eukaryotes have multiple RNA Pols and more complex control sequences

27
Q

Describe Eukaryotic RNA Pols;

  • RNA Pol l
  • RNA Pol ll
  • RNA Pol lll
  • others
A
  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
Q

What do eukaryotes use instead of the sigma factor to recognise different promoters?

A
  • accessory proteins
  • e.g. transcription factors
  • more complex and diverse than sigma
29
Q

Discuss RNA Pol ll (Eukaryotic);

  • constitutively v selectively transcribed
  • TATA box
  • CCAAT box
A
  • 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
Q

What are 3 important sequence elements in a eukaryotic gene?

A
  • TATA box: binds TBP
  • CAAT: binds CTF
  • GC: binds Sp1
31
Q

Discuss other regulatory elements: What, who where?

A

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
Q

What is post-transcription RNA processing why is it needed?

A
  • no precise Ter sites as in bacterial
  • allows for post-modification process to acquire biological activity
  • mRNA, tRNA, rRNA
33
Q

What happens in mRNA processing?

  • 5’ capping
  • 3’ polyadenylation
  • splicing
A

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
Q

Discuss splicing

  • enzyme used
  • alternative
A
  • 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
Q

What is meant by the genetic code?

A

correspondence between nucleic acid and polypeptide sequences

36
Q

What are codons?

  • how many bases per?
  • how many different triplets?
  • what are reading frames? how are they properly read?
A
  • 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
Q

Why is the genetic code degenerate and non-random? Give an example using start and stop codons.

A
  • 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
Q

Discuss tRNAs;

  • purpose
  • length
  • structure (acceptor stem and anticodon loop)
A
  • 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
Q

What occurs in tRNA Aminoacylation? (2 steps + enzyme involved)

A
  • aaRS appends aa to tRNA
  1. aa + ATP reaction activated aa into aminoacyl-AMP
  2. aminoacyl-AMP + tRNA = aa-tRNA
40
Q

How does an aaRS recognise a tRNA so that it can be charged with the correct amino acid?

A
  • aaRS can recognise unique structural features
  • proof-reading mechanism aids fidelity
41
Q

What are 6 functions of the ribosome?

A
  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
Q

Give an overview of Ribosome structure

A
  • 30s small subunit, 50s large subunit
  • A, P, E-sites
43
Q

Compare the small and large subunit of the bacterial ribosome?

A

30s - small
- 16 rRNA
- 21 proteins

50s - large
- 5s and 23s rRNA
- 31 proteins

44
Q

Discuss the 3 main sites on ribosomes that tRNA bind to.

A

A-site: aa-tRNA
P-site: peptidyl tRNA
E-site: deacylated tRNA

45
Q

Compare the eukaryotic ribosome and bacterial ribosome?

A

bacterial = 70s
eukaryotic = 80s

46
Q

Discuss the small and large subunits in rat liver cytoplasmic ribosome.

A

40s small
60s large

47
Q

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?
A
  • N to C terminus
  • chain is added to new aa
  • 5’ –> 3’
  • on polysome, ribosome binds to mRNA –> polyribosome
48
Q

How are peptide bonds formed with reference to the ribosomal peptidyl transferase reaction?

A

peptidyl-tRNA in P-site, added onto aa-tRNA in A-site

49
Q

In bacterial systems, how does it know that AUG codes a start and not a regular Met aa?

A
  • Shine-Dalgarno sequence
  • approx 10 baes upstream
  • ribosomal binding site

** Not in Eukaryotic

50
Q

Discuss chain initiation in bacteria.

A
  • 2 ribosomal subunits assemble with fMet-tRNA on mRNA
  • requires initiation factors (IF-1, IF-2, IF-3, in E.coli)
51
Q

Discuss chain elongation in bacteria + draw a diagram;

  1. Decoding
  2. Transpeptidation
  3. Translocation
  • the rate that this occurs
A
  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
Q

What are the 3 elongation factors?

A

Tu
Ts
G

53
Q

What is post-translational processing?

A
  • 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
Q

How are antibiotics useful in undersanding ribosomal mechanisms?

A
  • most block translation
  • by blocking a process, allows more detailed analysis
55
Q

Discuss streptomycin.

  • drug class
  • low conc. consequences
  • high conc. consequences
A
  • 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
Q

Discuss chloramphenicol.

  • drug class
  • cell activity
  • clinical use
A
  • 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
Q

Discuss Rifampicin.

A
  • 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

Bioc2201 (8 decks)

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