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Gene Regulation and Expression > Translation > Flashcards

Translation Flashcards

1
Q

What is Translation?

A
  • protein ( polypeptide) synthesis
  • nucelotide sequence information in DNA copied ( transcribed) into mRNA
  • the mRNA like strand of the DNA is the coding strand
  • the information in the mRNA is translated into the amino acid sequence of the corresponding polypeptide
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2
Q

How is translation carried out ?

A

-by the ribosome
( mRNA is read 5’ to 3’ direction)
- protein is synthesised from N- to C- terminus

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3
Q

What is the genetic code?

A
  • specifies which mRNA nucleotide sequence corresponds to which amino acid in a protein
  • the genetic code consists of non- overlapping triplets which are read from a fixed starting point ( francis crick)
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4
Q

what are the bases?

A
  • AGCT(U)
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5
Q

how many different amino acids are there ? and therefore how many different varieties of polypeptides are there?

A
  • there are 20 amino acids
  • 1 base per amino acid - 4 possible code words
  • 2 base per amino acid - 16 possible code words
  • 3 base per amino acid - 64 possible code words
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6
Q

Cracking the genetic code?

A
  • evidence against overlap or bracketing of code words ( codon)
  • single base changes in genes = single amino acid changes in protein
  • frameshift mutations affect the whole downstream sequence (i.e. no gaps /bracketing )
  • there was evidence that triplet (3base) codons form from synthetic mRNAs translated in vitro.
  • the code is uniersal- with a few excceptions where ther are limited cchanges ( the genetic code is identical in all organisms)
  • main exception = mitochondria
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7
Q

what is triplet binding assay?

A

it tetsts which synthetic trinucleotides can promote binding of specific tRNAs to ribosomes
- tRNAs mediate the interaction of amino acids with the genetic code.

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8
Q

What is tRNA?

A
  • transfer RNA
  • it acts as an adapter molecultes
  • processed from longer precursor RNAs
  • it bridges the gap between codon and amino acid
  • each tRNA has a triplet anticodon that recognises one orr more codons in the mRNA by base pairing
  • tRNAs bring the correct amino acid to the ribosome in response to a specific codon
  • the amino acid is added to the growing polypeptide,

HAS UNIVERSAL ‘ CLOVERLEAF ‘ STRUCTURE - defined by 4 stems and 4 loops ( the stem and leaf are called an arm)
- base pairs differ - depending on tRNA

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9
Q

What is the tRNA structure?

A
  • secondary structure : - maintained by invariant base pairs
  • tertiary structure:- L-shaped ; maintained by tertiary hydrogen bonds between the invariant bases

tRNAs need specific features (e.g. for charging enzymes)

tRNAs need common features - e.g. all bind to same site on ribosomes.

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10
Q

What do we know about tRNA base modifications?

A

tRNA’s are the most heavily modifieed post transcriptiional RNA
- tRNAs have may post - transcriptionally modified bases.

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11
Q

What is the role of tRNA base modifications?

A
  • to make the binding of different aminoacyl-tRNAs to both the A and P sites of the ribosome uniform
  • without the mpdifications - the ttRNA sequence and the particular amino acid linked to the tRNA affect the binding affinity for the two principal tRNA binding sites on the ribosome
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12
Q

What is anticodon modifications?

A
  • modified bases in the anticodon can affect the nature and efficiency off base pairing with codons.
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13
Q

what is meant by tRNA charging ?

A
  • covalent linkage of the tRNA 3-end to the cognate( means as specified by the anticodon) amino acid

Catalysed by an aminoacyl-tRNA synthetase

  • one enzyme per amino acid
  • each recogniises all isoaccepting tRNAs
  • linkage is via an ester bond to the 3’ - OH group
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14
Q

What is the two step - mechanism of tRNA charging reactions?

A

alpha alpha + ATP&raquo_space; aa ~ AMP + PPi

2.

alpha alpha~AMP =tRNA&raquo_space; aa ~ tRNA + AMP

( refer to slide 20 to get a better understanding !)

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15
Q

Why is accuracy of charging critical?

A
  • mischarged tRNAs lead to errors in proteins
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16
Q

How is accuracy mantained ?

A
  • it is controlled by the aminoacetyl - tRNA synthetases
  • each recognises all itss isoaccepting tRNAs by conntacts on the acceptor stem and anticodon loop
  • contacts aligned on one face of the tRNA
  • usually at least one anti-codon base is recognised
  • single base changes in the acceptor stem change the tRNAs identity
  • isoaccepting tRNA molecules contain an invariant discriminator ( present in all tRNAs to encode amino acids)base in the acceptor stem that controls their identity
17
Q

Speed versus accuracy ?

A

proofreading as a mechanism to avoid trading off one for the other !
- both amino acid selection and tRNA selection are subjecct to proofreading (1.e. error checking mechanisms )

  • proofreading either difavours the forward reaction ( kinetic proofreading) or reverses the catalytic reaction ( chemical proofreading) is the wrong component has been selected.
  • avoids the need for absolute accuracy (slow)
18
Q

another way that accuracy is maintianed is amino acid selection- what do you know about that?

A

Amino acid selection:
- chemical proofreading occurs on binding the tRNA - either the incorrect aminoacyl adenylate is hydrolysed or tRNA mischarging occurs followed by hydrolysis.

(e.g. Ile and Val are very similar)

19
Q

What do the active site and editing site have in relation to maintaining accuracy?

A
  • active site = excludes too large amino acids
  • editing site- can accept ones that are too small!

active and editing site form double molecular sieve
- conformational change in the acceptor stem of the tRNA moves the amino acid from the catalytic site to the editing site.

20
Q

What is proofreading and why is it important ?

A
  • tRNA selection - kinetic proofreading
  • correct tRNA binding induces a rapid conformational change - stabilising binding to allow fast aminoacylation
  • incorrect tRNA fails to induce the conformational change - allows tRNA time to dissociate before being mischarged,
  • the right tRNA produces rapid translation - the wrong one does not -so gives time to sort it !
  • mutation affects the accuracy of the enzyme - the enzyme has normal level of expression, activity , solubility and normal structure.
  • mutation is in editing site of enzyme
  • defect is proofreading - leads to higher levels of mis-charging with serine.( results in mix up of serine and alanine )
  • the experiment with a mouse ( causing neurodegenerative condition) shoes the need for quality control of tRnA charging !
21
Q

What is decoding?

A
  • refers to codon- anticodon recognition ( occurs on ribosome)
  • by base pairing between the codon and anticodon
  • ‘wobble’ and modified bases allow one tRNA to decode more than one codon
22
Q

What is the wobble hypothesis?

A

this is an extension to the normal rules of base pairing !
- watson - crick base pair rules between anticcodon position 1 and codon position 3 are relaxed due to flexibilty of the anticodon loop !

  • wobble rules explain how a single tRNA can decode a codon pair
  • also shows that unique codons must end in G or U because A and C cannot have a unique meaning !
  • modified bases restrict or enhance base- pairing in the wobble position.
23
Q

What is a summary of Decoding?

A
  • multiple tRNAs can recognise the same codons
  • single tRNAs can recognise multple codons
  • Wobble rules define a minimal set of 31 tRNAs plus an initiator tRNA for translating the universal genetic code.

( a special tRNA is required for protein synthesis)

Gene Regulation and Expression (10 decks)

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