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BIOL253: Genetics > Translation > Flashcards

Translation Flashcards

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1
Q

Describe the genetic code

A

mRNA arranged into triplet sequences called codons, which each encode an amino acid (61 amino acid coding, 3 stop codons)

  • universal
  • degenerate (every amino acid, except Trp and Met (AUG start codon) are encoded by more than one codon)
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2
Q

What are the three types of RNA involved in translation and what is their roles?

A

mRNA - encodes protein

rRNA - forms part of the ribosome used to translate mRNA into proteins

tRNA - couples region of ribosome which binds the mRNA codon and the amino acid

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3
Q

Describe the structure of a tRNA molecule

A
  • approximately 70 nt in length
  • forms a 2D clover-leaf structure with three hairpin loops due to regions of self-complementarity: T loop, D loop and anticodon loop.
  • folds into a 3D L-shaped structure due to base pairing between the T and D loops.
  • 3’ CCA single stranded tail located on the acceptor stem (usually added post-transcriptionally) is the site of amino acid attachment
  • anticodon loop contains an anticodon
  • tRNA contains unusual bases produced by editing (pseudouridine, dihydrouridine)
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4
Q

What are the steps involved in the attaching of a tRNA molecule to its cognate amino acid?

A

called charging or aminoacylation

1) amino acid activated by ATP hydrolysis and attachment of AMP to the carboxyl end of the amino acid
2) liberation of AMP as amino acid is carboxyl end is attached to the 2’ or 3’ hydroxyl of ribose of terminal adenine nucleotide in the CAA sequence

reaction catalysed by aminoacyl-tRNA synthetases (ATP cofactor, produces aminoacyl-tRNA)

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5
Q

How are aminoacyl-tRNA synthetases specific for tRNA and amino acid?

A

Editing (pre- and post-transfer) and size exclusion

  • one aminoacyl-tRNA synthetase enzyme for each amino acid (at least 20 different enzymes) each with a binding site specific for the amino acid and a binding site specific for the anticodon

Size exclusion:
- large amino acid cannot enter the amino-acylation site of a synthetase specific for a small amino acid

Editing pre-transfer:
- activated amino acid binds to amino-acylation site but is incorrect so enters the editing site where it is hydrolysed.

Editing post-transfer:
- activated amino acid binds to amino-acylation site and is transferred to the bound tRNA, is able to move into the editing site so is hydrolysed

(a correct amino acid will not be able to enter the editing site so will be released as a complete aminoacyl-tRNA)

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6
Q

What are the two types of base pairing that occur between the codon and the anticodon?

A

Watson and Crick base pairing

Wobble base pairing (can occur between the first nucleotide of the anticodon and the third nucleotide of the codon = provides part of the basis for degeneracy of genetic code)

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7
Q

If the first base of the anticodon is : C, A, U, G, or I, what are the bases in the third position of the codon that can pair with it via wobble base pairing?

A

Anticodon: Codon:
C G
A U
U A or G
G U or C
I U, C, or A

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8
Q

How does inosine nucleotide base occur?

A

hydrolytic deamination of adenine

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9
Q

What is normally always the first amino acid (although it may be removed later)?

A

methionine

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10
Q

What is the name of the specific tRNA molecule that is used for initiation involving methionine?

What specifically identifies this initiator tRNA?

A

Prokaryotes = tRNAfmet (methionine enzymatically formylated in bacteria)

Eukaryotes = tRNAimet

Identified by:

  • three G-C base pairs in the anticodon loop
  • lack of base pairing at the 5’ end within the acceptor stem (C-A mismatch in bacteria, A-U mismatch in eukaryotes)
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11
Q

What is the name of the specific tRNA molecule used for elongation involving methionine?

A

tRNAmmet

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12
Q

Describe the structure of the bacterial ribosome

A

bacteria have a 70s ribosome formed of a large subunit and a small subunit:

Large 50s subunit:

  • two rRNA molecules: 5s and 23s rRNA
  • contains about 34 proteins

Small 30s subunit:

  • one rRNA molecule: 16s rRNA
  • contains about 21 proteins
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13
Q

Describe the structure of the eukaryotic ribosome

A

eukaryotes have a 80s ribosome formed of a large subunit and a small subunit (bacteria and eukaryotes have similar overall ribosomal structure - conserved)

Large 60s subunit:

  • three rRNA molecules: 5s, 5.8s, and 28s rRNA
  • contains around 49 proteins

Small 40s subunit:

  • one rRNA molecule: 18s rRNA
  • contains around 33 proteins
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14
Q

What is the rate limiting step of translation?

A

initiation

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15
Q

Describe the initiation of translation in bacteria

A

Small ribosomal subunit has IF1 and IF3 in A and E site, respectively:

  • small ribosomal subunit binds to the mRNA: Anti-Shine-Dalgarno sequence of the 16s rRNA molecule binds to the Shine-Dalgarno (SD) sequence immediately upstream of start codon
  • tRNAfmet binds to the AUG codon in the P site of the small ribosomal subunit.
  • IF2 (with intrinsic GTPase activity) bound to GTP binds to IF1
  • once all components are bound, IF2 hydrolyses GTP to GDP and all the initiation factors are released and the large ribosomal subunit can associate (the amino acid of the tRNAfmet is within the exit tunnel of the large ribosomal subunit
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16
Q

What is the purpose of IF1 and IF3?

A

bind to the A and E site of the small ribosomal subunit, respectively:

  • ensure the tRNAfmet binds in the P site only
  • prevents binding of the large ribosomal subunit
17
Q

What are the three sites within the ribosomal complex?

A

A (aminoacyl site)
P (peptidyl site)
E (exit site)

18
Q

Describe the process of elongation?

A
  • next tRNA is recruited by EFTu (which has GTP bound and has intrinsic GTPase activity)
  • if the codon-anticodon pairing is correct, EFTu GTPase activity is activated hydrolyses GTP, released tRNA and dissociates from the ribosome
  • the CAA tail with the amino acid attached moves into the P site and peptidyl transferase forms peptide bond –>
  • A loop of large subunit rRNA binds to the amino acid of the aminoacyl tRNA
  • P loop of large subunit rRNA binds to the CAA tail of the peptidyl tRNA
  • -> positions for nucleophilic attack from the amino group of the aminoacyl tRNA amino acid to the carboxyl group of the peptidyl tRNA amino acid resulting in formation of peptide bond
  • EFG bound to GTP competes with EFTu:tRNA for binding in the A site.
  • hydrolysis of GTP (by EFG intrinsic GTPase activity) causes a conformational change in EFG and the ribosome forcing the movement of the ribosome three bases along in the 3’ direction and forcing the movement of the peptidyl tRNA from the A site into the P site, and the deacylated tRNA into the E site.
  • insertion of aminoacyl-tRNA at the A site dislodges the uncharged tRNA from the E site
19
Q

How can EFG bind in the A site?

A

it is a structural mimic of EFTu:tRNA

20
Q

How is EFTu regenerated following hydrolysis of GTP?

A

A guanine nucleotide exchange factor (EFTs) will exchange the GDP for GTP to reactivate the EFTu for re-use

21
Q

What is peptidyltransferase?

A

Carries out the peptidyl transferase reaction

  • not a protein, but is a ribozyme (an RNA section of the 50s/large subunit.
22
Q

Describe the process of translation termination in prokaryotes

A
  • stop codon is reached so no tRNA available to enter A site
  • Release Factor 1/2 binds to the stop codon
  • -> RF1 binds to UAA and UAG stop codons
  • -> RF2 binds to UAA and UGA stop codons
  • results in hydrolysis of the aminoacyl bond between the peptide chain and the peptidyl tRNA (leaving an uncharged tRNA in the P site)
  • RF3 bound to GTP binds to the large subunit of the ribosome causing release of polypeptide and formation of hybrid state tRNA, following intrinsic GTPase activation this causes the dissociation of the RF3 and RF1/2
  • the uncharged tRNA in the P site acts a signal for recycling to occur
  • -> ribosome recycling factor (RRF) recruited by EFG bound to GTP to the A site of the ribosome and force dissociation of the large and small subunits
  • -> uncharged tRNA released and IF3 binds to the E site to prevent the two subunits re-associating
23
Q

How is transcription and translation coupled in prokaryotes?

A

polycistronic mRNA is bound by multiple ribosomes synthesising protein simultaneously (polyribosome)

24
Q

What assists the binding of the ribosome to the mRNA in eukaryotes?

A

the 7-methylG 5’ cap

no Shine Dalgarno sequence

25
Q

What is the Kozak sequence?

A

RXXAUGG

- the preferred context for the start codon in mammals

26
Q

True or false: all of the eukaryotic mRNA acts as a template (is coding)? why/why not?

A

False:

- there are 5’ and 3’’ untranslated regions (UTRs)

27
Q

Describe the grouping of the eukaryotic initiation factors (eIFs)

A

12 eIFs numbered 1 to 6
- each number is a group of different types that are further named with a letter

(E.g. eIF4A, eIF4B)

28
Q

Describe the formation of the pre-initiation complex for translation in eukaryotes

A

1) eIF4E recognises and binds to the 5’ cap
2) poly-A-tail is bound by PABPs (Poly-A-binding proteins)
3) eIF4G (scaffold protein) binds to PABP and eIF4E to form a closed loop complex (connection between 5’ and 3’ end of mRNA)
4) eIF1A and eIF1 bind to the A and E site of the small ribosomal subunit, respectively. tRNAmet (initiator tRNA) recruited by eIF2 (intrinsic GTPase) is already bound to the P site on the small subunit along with eIF5 and eIF3.
5) eIF3/eIF4G interaction recruits the prepared small ribosomal subunit to the mRNA to form the pre-initiation complex.

29
Q

How does the pre-initiation complex of translation in eukaryotes locate the start codon?

A

eIF4A/4B has ATP dependent helicase activity, drawing the mRNA through until it reaches the start codon (AUG)

30
Q

Following location of the start codon, what are the events the proceed? (eukaryotes)

A

1) anticodon of tRNAmet binds to the start codon, resulting in activation of eIF2 GTPase activity of eIF2, causing a conformational change
2) recruitment of large subunit by eIF5B (a GTPase)
3) activation of eIF5B GTPase activity, causes release of majority of eIFs but closed loop complex maintained (eIF4E and eIF4G still bound to enhance the translatability of mRNA)

31
Q

What are the equivalent eukaryotic elongation factors to the bacterial elongation factors used in translatiom?

A

Bacterial Eukaryotic
EFTu eEF1A - binds aminoacyl tRNA and GTP
EFTs eEF1B - guanine nucleotide exchange
EFG eEF2 - ribosome translocation

32
Q

Describe the process of translation termination in eukaryotes

A

1) eRF3 (intrinsic GTPase) recruits eRF1 (release factor) recognises all codons and is a tRNA structural mimic - binds to A site at stop codon
2) GTPase activity of eRF3 causes it to dissociate from ribosome (eRF1 still bound in A site)
3) ABCE1 ATPase binds to eRF1 and hydrolyses the aminoacyl bond between the peptide chain and the peptidyl tRNA leaving an uncharged tRNA in P site (signal for recycling)
4) Hydrolysis of ATP by ABCE1 ATPase activity result in dissociation of the ribosomal subunits and the binding of eIF1, eIF1A and eIF3 to the small subunit .

33
Q

What is the main use of protein synthesis inhibitors?

A

Antibiotics:

- specific to the prokaryotic 70s ribosome (doesn’t inhibit eukaryotic 80s ribosome)

34
Q

Why do antibiotics have the potential to affect mirochondria?

A

mitochondria also have 70s ribosomes (similar to prokaryotic)

35
Q

Give three examples of antibiotics, their molecular targets, and their uses?

A

Tetracycline = binds to A site of 30s subunit and prevents recruitment of charged tRNA

Puromycin = mimics aminoacyl-tRNA and binds to the A site and peptidyltransferase centre transfers the polypeptide chain to the puromycin moiety and polypeptidyl-puromycin is released from the ribosome
- broad spectrum

Fusidic acid = binds to EFG to prevent the release of EFG-GDP from the ribosome, inhibiting the translocation of the ribosome and blocks the A site for next tRNA.
- narrow spectrum - used to treat Staphylococci (E.g. MRSA) and structural studies of ribosome function

36
Q

Describe how the diphtheria toxin inhibits translation in eukaryotes

A

produced by Corynebacterium diptheriae
- catalyses the ADP-ribosylation of eEF2 using NAD+ as a cofactor to inactivate eEF2 and block the binding of another tRNA

37
Q

How does Ricin affect translation in eukaryotes?

A

a type 2 ribosome inactivating protein (RIP)

  • initially synthesised as a prepro-polypeptide containing both A and B chains
  • then split by proteolysis but still linked by disulphide bridge

Chain A
- an RNA N-glycosidase that binds and depurinates adenine of the 28s rRNA, which is then hydrolysed

Chain B
- a lectin glycoprotein that binds galactose, allowing it to enter the cell through the membrane and endoplasmic reticulum

BIOL253: Genetics (6 decks)

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