Cell Biology- Hejmadi5-6- DNA translation

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Give 3 key features of the genetic code
1.Triplet code (3 bases = 1 amino acid) 2.Degenerate (more than 1 triplet may encode the same amino acid) 3. Non overlapping (always read in complete blocks- doesn’t overlap with preceding codon)
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Instead of AUG, start codons can rarely be...? What does this code for?
GUG- valine
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Give the 3 universal stop codons
UAG, UGA, UAA
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There are some exceptions. What does a)UGA code for in mitochondria b)CUG code for in yeast mitochondria c)UAA and UAG in paramoecium
a)Trp b)Thr c)Gln
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What is the adaptor hypothesis?
postulates that the genetic code is read by molecules that recognise a particular codon and carry the corresponding amino acid. (tRNA)
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What is a tRNA molecule?
a class of small RNAs which form covalent bonds to amino acids, allowing correct insertion of amino acids into the elongating polypeptide chain.
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Describe the structure of tRNA molecule
A ‘cloverleaf’ structure with 5’ phosphate end and a 3’ hydroxyl end. The structure consists of a 7bp stem, a D-arm, an anticodon arm, a T arm, and a variable arm.
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Why are loops formed?
Because it contains non-watson-crick base pairing... e.g. GU in 7bp stem- there are no hydrogen bonds between these pairs.
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Describe the 7bp stem. What else is it known as and why?
Includes the 5’ terminal nucleotide and may include non Watson-Crick base pairs such as GU. Known as the acceptor/amino acid stem as this is where amino acid residue is carried.
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Describe the D-arm
A 3 or 4bp stem ending in a loop (from 5-7 nucleotides) that frequently contains the modified base dihydro-urodine. Stem and loop are collectively known as D arm.
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Describe the anticodon arm
A 5bp stem ending in a loop that contains the anticodon- the triplet of bases that are complementary to the codon on mRNA
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Describe the T-arm
A 5bp stem ending in a loop that usually contains the sequence T Ψ C.
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What does the symbol Ψ represent?
pseudo-uridine
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Describe the variable arm
This is the site of greatest variability among tRNAs. It has from 3 to 21 nucleotides and may have a stem consisting of up to 7bp. It is found between the anticodon loop and the T loop.
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Why are there more tRNAs than amino acids?
due to the degenerate nature of the genetic code. Therefore there can be tRNAs with different anticodon sequences carrying the same amino acid.
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What does tRNA "charging" refer to?
The addition of the correct amino acid to the tRNA molecule
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What enzyme catalyses this reaction? Describe it.
aminoacyl tRNA synthetase. (aaRS). It has two sites- one for the binding of tRNA and one for the binding of amino acid.
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What type of reaction is this and what joins together?
a transesterification reaction where the 3’-OH end of tRNA is linked to the carboxyl end of amino acid
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What 2 things does the enzyme therefore do?
1.Selecting correct tRNA to be acylated -by recognizing specific identifiers present on acceptor stem & anticodon loop 2. Selecting the correct charged tRNA as specified by mRNA. -Ribosomes select charged tRNA from their codon-anticodon interactions
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What is the wobble hypothesis?
The 1st 2 codon-anticodon pairings have normal WC geometry. →creates structural constraints ensuring that 3rd pairing can not drastically change conformation from that of a WC pair. BUT 3rd codon can allow limited adjustments in pairing geometry
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What does this permit?
the formation of several non-Watson-Crick pairs
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Which three 5' anticodon bases on RNA can form non-watson crick base pairs with 3' codon bases on mRNA?
1) U can bind to A or G 2)G can bind to C or U 3)I can bind to U, C or A
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What does this account for? Therefore?
codon degeneracy. Therefore one tRNA can translate more than one triplet code on mRNA.
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How many different tRNAs are required to translate all 61 codon triplets of the genetic code
At least 31
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What are ribosomes made up of? What do they consist of?
made up of ribosomal RNA and ribosomal proteins, and consist of 2 subunits; large and small.
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What is the large subunit made up of?
49 or more proteins + 3 RNA molecules
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What is the small subunit made up of?
82 proteins + 4 RNA molecules
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What is special about these subunits?
They are self-assembling, and combine only in the presence of mRNA and a charged (aminoacylated) tRNA.
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Translation requires what 3 things. What do they together create?
Charged tRNA, ribosome and mRNA. They create 3 sites known as APE.
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What is the A site?
aminoacylation site (arrival site)
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P site?
protein synthesis site
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E site?
Exit site
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What are the 3 distinct steps of polypeptide synthesis?
Chain initiation, chain elongation, chain termination
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What does eIF stand for?
Eukaryotic Initiation factor
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What is a KOZAK sequence?
A highly conserved sequence - ACC AUG G -contains start codon
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Which part is variable and which is constant?
The CC is variable, the A and G is always constant, therefore it provides a shape that a protein complex can recognise when looking for correct start codon
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Describe step 1 and 2 of chain intiation
1)binding of eIF3 and eIF1A to 40S subunit in the inactive 80S ribosome, which then releases the 60S subunit. 2)The ternary complex of eIF2, GTP and Met-tRNA bind to the 40S ribosomal subunit accompanied by eIF1 to form 43S pre-initiation complex.
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Describe step 3, 4 and 5
3) mRNA is activated by eIF4F complex, eIF4B and ATP. 4)43S Preinitiation complex attaches to mRNA (Cap binding) 5) initiation complex unwinds mRNA using eIF4 helicase stopping at the start site AUG
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What does the recognition of this site cause? (step 6)
an irreversible GTP hydrolysis of eIF2 preventing any further unwinding, and the 48S preinitiation complex is formed.
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What does this mediate the association of? (step 7-8)
60S-eIF6 (large subunit) to the small subunit by the action of eIF5. The 80S initiation complex is formed with a P site.
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What is the first step of chain elongtion?
charged tRNA binding (i.e. decoding)
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When does elongation of amino acid chain proceed?
Once an initiator tRNA is positioned at the AUG start codon
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Describe the charged tRNA binding
A binary complex of GTP with the elongation factor EF1A combines with an aminoacyl-tRNA. The ternary complex formed binds to the ribosome and the aminoacyl-tRNA is bound in a codon-anticodon complex to the ribosomal A site
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What is the second step of chain elongation?
Conformational change in ribosome
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Describe conformational change
The GTP is hyrdolysed and hence EF1A-GDP + Pi released from the complex. This leads to a physical change in terms of where the amino acids are. The change in shape allows amino acid 2 (at the A site) to be pushed closer to the amino acid 1 (P site)
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What is step 3?
Transpeptidation
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Describe transpeptidation
Peptidyl transferease (located on large subunit of ribosome) catalyses peptide bond formation. The peptide bond is formed between nh2 group from amino acid 2 (site A) and the carboxyl grp of first amino acid (site P).
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What is step 4?
Translocation (ribsosome moving to next codon)
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Describe it
the now uncharged P site tRNA shifts to the E site on the ribosome. The ribosome translocates the length of one codon (i.e. shifts across to next codon), releasing the empty tRNA.
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What is happening simultaenously?
the peptidyl-tRNA in the A site, together with its bound mRNA is moved to the P-site.
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What does this process require the participation of?
elongation factor EF2, which binds to the ribosome with GTP and is only released on hydrolysis of GTP to GDP+ Pi
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What release factors recognise and bind to stop codons?
eRF1/2 and erF3-GTP
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Which release factors recognise which stop codons?
eRF1 recognises UAA and UAG eRF2 recognises UAA and UGA
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What did these release factors induce?
peptidyl transferase to transfer peptidyl group to water instead of aatRNA.
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What happens next?
Uncharged tRNA is released from ribosome, as well as eFR1 and eFR3-GDP + Pi. Inactive ribosome then releases mRNA.
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Typically, how long does the entire translation process take?
30-60seconds
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How does the circular shape of mRNA increase translational efficiency?
Circular mRNA = more efficient use of space.Therefore can fit lots of mRNA in small space, hence enabling more translation
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Name 3 post translational modifications
1) Protein folding 2)Covalent modification 3)proteolytic cleavage
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Describe protein folding
Nascent protein is folded and/or modified Mediated by molecular chaperones (e.g. Hsp70) or chaperonins (Hsp60 complexes)
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Describe covalent modifications
Addition of side groups (e.g. acetyl, phosphoryl, hydroxyl, glycosyl etc.) Dictate the activity, life span or location of proteins
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Describe Proteolytic cleavage
Activates some inactive precursors Most common type of post-translation modification- usually involving the removal of their leading Met residue
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Give an example of proteolytic cleavage.
if protease was syntehsised as an active protein, it would digest all proteins inside the cell! →synthesised as inactive instead and only under certain conditions are they activated through proteolytic cleavage.
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Instead of AUG, start codons can rarely be...? What does this code for?

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GUG- valine

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Give the 3 universal stop codons

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Card 4

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There are some exceptions. What does a)UGA code for in mitochondria b)CUG code for in yeast mitochondria c)UAA and UAG in paramoecium

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Card 5

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What is the adaptor hypothesis?

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