MCG - (Worksheet) Transcription, Translation and DNA Analysis Flashcards
Heteronuclear RNA (hnRNA) is modified in several ways in its conversion to mRNA.
Give examples of two of these modifications and explain their functional significance.
Organisation of DNA in eukaryotic genes includes exons and introns (non-coding regions). The introns need to be removed and the exons spliced together to give the correct sequence of triplet codons for the sequence of amino acids in the final protein.
Capping at the 5’ end with 7-methyl guanine: promotes binding of mRNA to the ribosome, prevents attack by nucleases
Addition of the polyA tail at the 3’ end: aids exit of mRNA through the nuclear pores and confers stability to mRNA in the cytosol.
Briefly summarise the structural differences between tRNA, mRNA and rRNA.
tRNA:
- 75 – 90 nucleotides long
- folds to clover leaf 3-D structure
- has aa binding site+ anti-codon
mRNA:
- codes for proteins
- single stranded
- very variable in length
- coding region starts with AUG,
rRNA:
- several components of variable size
- has considerable secondary structure with many hairpin loops
- associates with ribosomal proteins to form small and large ribosomal subunits
List the four stages of translation and indicate at which of these stages whether ATP or GTP is required.
- Amino acid activation to amino acyl t-RNA (needs ATP)
- Formation of initiation complex (needs GTP)
- Elongation stage (translocation step needs GTP)
- Termination - release of completed polypeptide chain (needs GTP)
Define the term ‘initiation codon’ and state what the triplet codon is.
It is the start codon, AUG, found at the 5’ end of coding region of every mRNA (and it codes for methionine in eukaryotes) .
Define the term stop codon and give one example.
It is the 3 triplet codons which cause dissociation of the finished polypeptide chain.
An example could be any of:
- UAA
- UAG
- UGA
Give one example of an amino acid that has one unique codon.
Methionine (AUG), or tryptophan (UGG).
Give one example of an amino acid that has 6 codons.
Any of:
- leucine
- arginine
- serine
Name the enzyme responsible for peptide bond formation during the elongation stage of protein synthesis.
State where the enzyme is located and indicate one unusual feature of the enzyme.
Peptidyl transferase, located on the large ribosomal subunit (recent experimental evidence suggests that its catalytic activity may be due to a region of RNA in the large subunit, not a protein-based enzyme).
The micro-organism responsible for diphtheria produces a toxin that blocks protein synthesis.
How does the toxin act and why does the patient feel unwell for such a long time?
The toxin produced by the diptheria micro-organism travels in the blood to other tissues such as the heart and kidneys.
The toxin covalently modifies the elongation factor required in the translocation step of protein synthesis, inhibits the synthesis of new proteins and thus causes tissue damage.
The toxin is very stable, and the anti-toxin given by injection only reacts with the free plasma form of the toxin, not the intra-cellular bound form.
Why do antibiotics inhibit protein synthesis in bacteria, but not in eukaryotic cells?
Several antibiotics inhibit protein synthesis in bacteria by interacting with the ribosome. The ribosome (50S and 30S subunits) in bacteria has a different structure to the ribosome in eukaryotes (60S and 40S). Antibiotics act specifically on the prokaryote ribosome and do not affect eukaryotic protein synthesis.
What is the mechanism of action for chloramphenicol?
inhibits the peptidyl transferase activity of the 50S ribosomal subunit (elongation stage)
What is the mechanism of action for tetracycline?
inhibits the attachment of aminoacyl t-RNA to the 30S ribosomal subunit (initiation stage)
What is the mechanism of action for rifamycin?
binds to RNA polymerase and prevents copying of DNA to mRNA (transcription)
What is the mechanism of action for puromycin?
puromycin resembles part of the structure of an amino acyl t-RNA, thus binds to the large ribosomal subunit, and causes premature termination of the growing polypeptide chain (termination stage)
Explain what is happening in the steps of PCR?
Step 1: The denaturing step.
The doubled stranded DNA template is denatured to two single strands of DNA by heating the reaction mixture to 95°C.
Step 2: The annealing step.
The forward primer and reverse primer anneal to their single stranded DNA templates typically at around 55°C (the exact annealing temperature is primer specific).
Step 3: The elongation step.
The Taq or Pfu polymerase (both thermophilic or heat loving polymerases) binds to the 3’ end of the primer and starts to add nucleotides (dATP, dCTP, dGTP and dTTP) to synthesise a new strand of DNA at 72°C.
