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BIOL2010 > Translation > Flashcards

Translation Flashcards

1
Q

What is translation catalysed by?

A

A ribozyme (the ribosome)

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2
Q

Is translation accurate?

A

Yes, theres a 1/10,000 error rate

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3
Q

What happens to DNA before translation?

A
  • The genetic message (DNA) is first TRANSCRIBED into mRNA
  • Transcribed from the alphabet of nucleotides in DNA, remains in the nucleotide “language” in mRNA [TRANSCRIPTION]
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4
Q

What is translation?

A
  • TRANSLATION is the process by which the genetic message, encoded in the sequence of the four RNA bases A, U, C and G is expressed in the form of an amino acid sequence in a protein using the 20 amino acids.
  • Translated from the language of nucleic acids into the language of proteins [TRANSLATION]
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5
Q

What does the amino acid sequence of a protein specify?

A

The amino acid sequence of a protein specifies the folding, structure and properties of the protein and is therefore dependent on the original DNA base sequence

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6
Q

What do we need for protein synthesis?

A
  • A template that has clues on where to start decoding
  • A supply of building blocks
  • Something to assemble the building blocks into chains
  • A way to supply the correct building block at the appropriate time
  • Rules to define HOW to decode (including where to start and stop)
  • Energy to drive the process
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7
Q

Tell me about the energy requirements for translation and where this energy comes from?

A
  • Ribosomes are the synthetic machinery (rRNA and protein components)
  • The template is a mature mRNA (capped and tailed in eukaryotes)
  • All 20 amino acids are activated as aminoacyl-tRNAs
  • Requires a large number of other enzymes/proteins (factors) and energy in the form of GTP and ATP
  • Adding each amino acid ‘costs’ 4x ATP
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8
Q

What is the general ribosome structure?

A
  • Small and large subunits have to join otherwise no translation
  • Association creates three sites for tRNA to occupy
  • mRNA slides through a channel which is on small subunit
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9
Q

What is each subunit in the ribosome for?

A
  • P= for peptidyl, is the second binding site for tRNA (holds a tRNA that carries a growing polypeptide)
  • A= aminoacyl, the first binding site for the ribosome (accepts incoming tRNA bound to AA)
  • E= exit (the tRNA goes here after its empty)
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10
Q

Whats the template for translation and what is it composed of?

A

mRNA

ribonucleotides (A, C, G and U)

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11
Q

What do mRNAs contain?

A

Non-coding or untranslated regions (NCRs, UTRs) at their 5’- and 3’- ends

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12
Q

What does the template of mRNA need to define?

A

Exactly where to start (determines ORF)

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13
Q

How many RNAs does the genetic code use and what for?

A

The genetic code uses three RNA bases to specify one AA (triple code)

Each of the 20 AA is specified by one or more triplets

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14
Q

What is the protein synthesised from?

A

The protein is synthesised from a continuous sequence of non-overlapping Codons running from an initiation codon to a termination codon

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15
Q

Whats are the stages to translation?

What does each stage require?

A
  1. initiation
  2. elongation
  3. termination
  4. recycling

Each stage requires other protein factors

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16
Q

Tell me about the initiation stage of translation?

A

Initiation (IFs/eIFs)

  • positioning of the small ribosomal subunit (and first aminoacyl-tRNA) at the initiation codon
  • joining of large ribosomal subunit to make whole ribosome
  • Initiation is the slowest step therefore limits rate of translation and is the step where most translational control occurs
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17
Q

Tell me about the elongation stage of translation?

A

Elongation (EFs- PROKARYOTES/eEFs- EUKARYOTES)

  • ensures correct amino acid is added sequentially to the growing protein chain by base pairing of transfer RNA (tRNA) with mRNA
  • decodes 10 – 40 aa per sec with only 1 in 10,000 error rate
  • Bring in the next tRNA
  • Join the second amino acid onto the first
  • Move along to the next triplet
  • Expel the first tRNA
  • Keep doing these cycles until…
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18
Q

Tell me about the termination stage of translation?

A

Termination (RFs/eRFs)

  • release of completed polypeptide when the stop codon is reached
  • No tRNA corresponding to the stop/termination codon
  • Bring in a factor to release the completed polypeptide
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19
Q

Tell me about the recycling stage of translation

A

Recycling (RRF in prokaryotes, ABCE1 in eukaryotes)

  • ribosomal subunits detach and are kept separated to allow new round of translation
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20
Q

Tell me about initiation in the protein synthesis in bacteria…

  • what does mRNA bind to?
  • Whats produced from this?
A
  • mRNA binds a special formylmethionine-tRNAf (only AA-tRNA to enter ribosome at P [peptidyl]-site + the 30S subunit at the P-site using initiation factors IF1, IF2 and IF3 and GTP.
  • This gives the “initiation complex”
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21
Q

Tell me about the initiation factors used in the initiation stage of protein synthesis in bacteria?

A
  • IF1 – binds in A site, prevents elongator tRNAs entering
  • IF2 – binds the GTP and the fMet-tRNAf
  • IF3 – prevents association with 50S, helps ensure fidelity of initiation codon selection (not present in all bacteria)
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22
Q

What do the sequences in bacteria mRNA help the ribosome do?

A

locate the initiation codon

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23
Q

What does prokaryotic mRNA possess?

What does this do?

A
  • A shine-dalgarno sequence that base pairs to the 3’ end of the 16s rRNA
  • This places the start codon AUG at the ribosome P-site about 10 bases 3’ of the S-D sequence
  • This is followed by the binding of the 50S subunit and dissociation of IF1 and IF3.
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24
Q

In protein synthesis in bacteria in the initiation stage, what happens to the IF1 and IF3 once the 30s is at the initiation codon?

What is this accompanied by?

A
  • it dissociates
  • This is accompanied by the binding of the 50S subunit and hydrolysis of the GTP bound to IF2, causing IF2 to also dissociate
  • This gives the 70S ribosome (50s + 30s) for elongation with the first tRNA in the P site, and an empty A site ready for the next tRNA
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25
Q

There are differences between eukaryotic and prokaryotic mRNA. Tell me some things that eukaryotic mRNAs contain?

A
  • a cap (made from a modified G nucleotide) and a poly(A) tail
  • No Shine-Dalgarno sequence
  • 5’ and 3’ UTRs may contain several features (sequence or structure-based) that help regulate protein expression/mRNA stability/localisation
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26
Q

In the initiation stage in eukaryotes, whats the role of the 5’- cap?

A
  • eIF3 equivalent to IF3, but 13 subunits
  • Binds eIF4G and the 40S subunit
  • PABP = poly(A)-binding protein
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27
Q

Eukaryotic initiation different considerably from bacterial initiation, how…?

A
  • Eukaryotic initiation differs considerably from bacterial initiation
  • eIF2 + the small 40S ribosome subunit + methionyl-tRNAimet + GTP bind to each other
  • The 40S subunit binds to mRNA with other initiation factors (eIFs) bound to the cap and poly A tail regions
  • The 40S ribosome then “scans” the mRNA looking for the AUG initiation codon usually uses first AUG it encounters
  • More efficient if this AUG is within the Kozak Consensus
  • eIFs then dissociate and the 60S subunit binds
  • Intact cap and tail regions are essential for initiation
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28
Q

What are the similarities between prokaryotic and eukaryotic initiation and the key differences?

A
  • 48S pre-initiation complex (PIC) formation and mRNA circularisation
  • 40s ribosomal subunit
  • 48s is the whole unit
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29
Q

mRNA transcribed in vivo is in combination with or without what?

A

Synergism between 5’ and 3’ ends

  • mRNA transcribed in vitro, in combinations with or without m7G cap or poly(A) tail
  • Added to rabbit reticulocyte lysate (contains ribosomes and other factors required for translation) in presence of radioactive methionine
  • Products of in vitro translation separated by SDS-PAGE and visualized with autoradiography
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30
Q

Without the eIF4E:eIF4G interaction, how can the ribosome attach to the mRNA?

A
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31
Q

Without the first tRNA, how do you start synthesising the polypeptide?

A
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32
Q

Function and control of the eLF2

A
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33
Q

Name some of the eLF2alpha kinases?

A
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34
Q

Activation of PKR in response to viral RNA…

What does this cause?

A
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35
Q

Protein targeting and export

A
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36
Q

Protein processing for secretion

A
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37
Q

How do many proteins fold?

A
  • Many proteins can fold spontaneously using only the primary amino acid sequence information
  • Other proteins require assistance from chaperones
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38
Q

Name some chaperones for protein folding?

A

Chaperones – e.g., heat shock proteins – prevent illicit liaisons between proteins (e.g., interactions between exposed hydrophobic regions)

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39
Q

What happens to malfolded proteins?

A

They are (hopefully) rapdily degraded

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40
Q

What does PERK exist for?

A

Protein misfolding in the ER can be a serious problem, and PERK exists to couple protein folding to protein synthesis

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41
Q

Tell me about the role of PERK in ER stress

A
  • Normally, BiP binds to PERK and keeps it in an inactive monomeric state
  • Can elicit ER stress using brefeldin A (blocks protein processing) or thapsigargin (interferes with ER Ca2+)
  • BiP dissociates to bind to unfolded proteins, activating PERK dimers
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42
Q

When PERK mice develop diabetes, what does this suggest?

A

Suggests important role for PERK in cells that secrete a lot of protein e.g. pancreatic b-cells

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43
Q

Whats Wolcott-Rallison disease [recessive]?

A

Loss of PERK function – patients develop Type I diabetes, growth retardation, multiple other effects

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44
Q

summary of first lecture

A
  • Translation is the synthesis of chains of amino acids into protein by decoding the message that was stored in the DNA (therefore change of “language”
  • mRNA is the intermediate, produced by transcription (nucleotide language remains similar)
  • Initiation is the positioning of the small ribosomal subunit at the initiation codon, and the joining of the large ribosomal subunit
  • Prokaryotic initiation uses rRNA:mRNA base pairing to define the start point, eukaryotic uses a sequence consensus
  • Eukaryotic initiation is more complex but more tightly controlled
  • Eukaryotic translation initiation is controlled by multiple mechanisms
  • Availability of cap-binding protein
  • Availability of ternary complex (contains first Met-tRNAi)
  • Proteins are sometimes also transported during translation
  • Accumulation of misfolded proteins causes activation of PERK
  • Next lecture – how amino acids are added to the correct tRNA
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45
Q

What are the building blocks for proteins?

A
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46
Q

What are the 20 proteinogenic amino acids?

A
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47
Q

What are the two types of amino acids and their subgroups?

A

There are polar and non-polar amino acids.

Polar- hydrophilic

  • Acidic: COO- provides the -ve charge
  • Basic: N+ provides the +ve charge
  • Uncharged polar: OH provides polarity

Non-polar- hydrophobic

  • R group usually contains just C and H
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48
Q

The 20 AA give properties that are essential for the formation of proteins

A
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49
Q

The genetic code is universal with some minor variations. What does this suggest?

A

This indicates that life arose from one original ancestor

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50
Q

Organisms exhibit codon usage bias. What is this and why is it important?

A

A preference to decode certain codons and not others

Important if making e.g. human protein in E. Coli

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51
Q

The genetic code is described as being what…?

A

triplet

continuous

non-overlapping

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52
Q

Given the nature of the genetic code, if one RNA base specified one amino acid, how many amino acids could be encoded?

A

If one RNA base specified one amino acid, only 4 amino acids could be encoded- one amino acid for each base

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53
Q

Given the nature of the genetic code,

if a pair of any of the four RNA bases specified one amino acid, how many amino acids could be encoded?

A

If a pair of any of the four RNA bases specified one amino acid, then only 16 amino acids (42) could be encoded.

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54
Q

Given the nature of the genetic code, a triplet of any of the four bases can specify how many combinations?

A

A triplet of any of the four bases can specify 64 combinations (43), therefore provides more than sufficient triplets to specify each of the 20 amino acids.

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55
Q

Why does the genetic code have to be continuous?

A

Addition or deletion of single nucleotide bases changes the frame downstream, therefore the code has to be continuous

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56
Q

Why can’t the genetic code be overlapping?

A

The continued contact of tRNAs with the mRNA template during elongation steps means that the code cannot be overlapping

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57
Q

The genetic code is degenerate, what does that mean?

A

That more than one codon may specify a given amino acid

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58
Q

3 of the AA have 6 codons each, what are these AA?

A

S, L and R

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59
Q

5 of the AA have 4 codons each, what are these AA?

A

G, P, A, T and V

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60
Q

Each of the four for any one AA starts with the same how many bases?

A

2

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61
Q

What have 3 codons each?

A

Isoleucine (I) and “stop”

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62
Q

9 AA have 2 codons each, what are these AA?

A

F, D, N, E, Q, H, C, Y and K

each pair either ‘ends’ with purines or pyrimidines

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63
Q

What is Methionine (M) specified by?

A

AUG

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64
Q

What is Tryptophan (W) specified by?

A

UGG

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65
Q

The degeneracy of the genetic code, allows for what?

A

silent mutations in DNA/mRNA (usually the third position of a codon)

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66
Q

What allows for conservative mutations to occur?

A

The arrangement of the code

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67
Q

question to think about: What is the consequence of a cytosine deamination mutation in each of the three bases?

A
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68
Q

codons

A
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69
Q

D614G missense mutation in SARS-CoV-2

A
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70
Q

Most tRNAs contain between how many bases?

A

70 and 90 bases

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71
Q

What do all tRNAs have?

A

highly conserved tertiary structures (L-shape) with much internal base pairing

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72
Q

What does the 3’-terminus of every tRNA end in?

How is each amino acid linked?

A

CCA

Each amino acid is linked as an ester to the 3’-OH (or in some cases to the 2’-OH) of the last A

73
Q

What do tRNAs contain?

A

Numerous unusual post-transcriptionally modified bases

tRNAs possess an anticodon that recognises the mRNA codon by anti-parallel base pairing

74
Q

Tell me about the tRNA structure?

A
75
Q

tRNA secondary structure e.g. yeast tRNAPhe, why is it methylated?

A

Its methylated so it doesn’t look like a foreign invader to the body

76
Q

Tell me about modified bases in tRNAPhe?

A

Pseudouridine (ψ) has more possibilities for H-bonding (a,d) and more rigidity

This is better for stability in tertiary structure and interaction with ribosome

77
Q

tRNAPhe

A
78
Q

What type of bond links amino acids and what does it synthesis require?

A

The peptide bond -CO-NH- links amino acids and it is an amide bond and its synthesis requires the input of energy

79
Q

How are AA linked to an tRNA?

A

Each of the 20 AA is first linked to the 3’-OH (or 2’-OH) of the terminal adenosine of a specific tRNA by an ester bond (gives an amino acyl-tRNA)

80
Q

What does the hydolysis of the ester bond help drive?

What does this ensure?

A

Peptide bond formation in the ribosome

This ensures an amino acid is only linked to its correct tRNA, highly specific enzymes are used

These vital enzymes are called amino acyl-tRNA synthetases

81
Q

Question: These enzymes ensure that a tRNA is linked to its correct amino acid so must show high specificity and fidelity – How?

A
82
Q

How are Amino-acyl tRNA synthetases produced?

A
  • tRNA synthetases recognise the tRNA and the amino acid
  • They catalyse the reaction of the terminal adenine ribose (of CCA) with the amino acid carboxyl group to give NH2CHRCO-3’O-tRNA
  • Two classes of synthetases exist (I and II)
83
Q

Activation of an amino acid occurs in a two-step process, what are these stages?

A
  1. Amino acid + ATP + Enzyme –> Enzyme-AMP-Amino acid + PPi

“activated intermediate”

(- = high energy bond)

  1. Enzyme-AMP-amino acid + tRNA –> Aminoacyl-tRNA + AMP + Enzyme
84
Q

During the activation of an amino acid, how many ATPs are used?

A

The equivalent of two ATPs is used (the enzyme uses a second to drive the reaction)

85
Q

What is the error rate for the activation of an amino acid?

A

1 in 10,000

86
Q

What does a two step reaction allow?

A

Proofreading- mistakes made in reaction 1 are eliminated in reaction 2

87
Q

Tell me about proofreading by amino acyl-tRNA synthetases e.g. tRSVal

A

in this example…

  • Phe cannot bind to the acylation site as it is to big
  • Alanine has managed to get into the acylation site as its small, however cannot bind to the hydrolytic site as the wrong tRNA has been made from when it bound to the acylation site so its not complementary
  • Val fits in both
88
Q

What is the anticodon essential for?

A

The anticodon plays no role in loading of correct amino acid onto the tRNA but is essential for correct translation of code

89
Q

The anticodon plays no role in loading of correct amino acid onto the tRNA but is essential for correct translation of code

Therefore, if an amino acid is linked to the wrong tRNA what happens?

A

then the amino acyl-tRNA still recognises the anticodon and inserts the wrong amino acid

90
Q

Experimental proof that the anticodon recognises the mRNA codon not the amino acid

A

Cysteine-tRNACys is prepared

91
Q

Tell me about Cysteine- tRNACys preparation…

A
  • Raney nickel (mix of Ni and Al) reduces cysteine’s -CH2-SH group into -CH3 (alanine)
  • Therefore cysteine-tRNACys is converted into alanine-tRNACys
  • The modified alanine-tRNACys is used in the synthesis of haemoglobin in an in vitro experiment
  • The result is that alanine is incorporated into the haemoglobin in place of each cysteine (UGU or UGC codons)
  • Thus, the anticodon is recognised, not the amino acid (alanine)
  • Therefore accuracy/proofreading in aminoacylation of tRNAs is critically important
92
Q

What does “wobble” mean?

A

Its a term coined by Crick that defines the ability of the first base of an anticodon to base pair with more than one codon

93
Q

What does a “wobble” allow?

A

An aminoacyl-tRNA to dock with more than one codon, so that fewer than 61 tRNAs are needed to decode the 61 codons

94
Q

What does wobble generally involve?

A

Wobble generally involved the nucleotides U or inoside (I) at the first position of the anticodon (matches 3rd position of codon)

95
Q

Tell me about the unusual base pairing in RNA

A

G pairs with C

A pairs with U

U can also pair with G

96
Q

Tell me the bases U can pair with and in what positions

A

U in the first anticodon position can base pair with A or G in the 3rd position of the codon

97
Q

Tell me what bases G can pair with and in what positions?

A

G in the first anticodon can base pair with C or U

98
Q

As G and U can pair with multiple bases the synonymous pairs of codons end U/C or A/G (Look at pairings in the codon table)

A
99
Q

There is further degeneracy of base-pairing in the first position of anticodon, what do some anticodons of tRNA have?

A

Inosine (I) in the 1st position (5’- position)

100
Q

What does Inosine (I) look like?

When is this made?

A

guanosine without an amino group but is made by deamination of adenine

This conversion is done AFTER transcription of the tRNA

101
Q

What can Inosine (I) pair with?

And with how many H bonds?

A

A, U or C using 2 H bonds

102
Q

Example of wobble binding: If the first base of the anticodon is inosine (I) then it can pair with what?
What does it mean it terms of tRNA recognition?

A

If the first base of the anticodon is inosine (I) then it can base pair with A, U or zc

Therefore, a single tRNA can recognise More then one codon

e.g. the 5’-IGG-3’ anticodon of tRNAPro can recognise three of the proline codons

103
Q

Summary of key roles of tRNA

A
  • To activate the amino acid as a tRNA ester
  • To act with the correct synthetase to ensure that the correct amino acid is linked to its tRNA
  • To base pair its anticodon triplet with the correct codon of mRNA - decoding role (anticodon:codon pairing can “wobble”)
  • To bring the correct amino acid to the ribosome
  • To facilitate peptide bond (-CO-NH-) formation of the nascent chain and the next amino acid
104
Q

Short answer question from 2016/2017 BIOL2010 exam:

Compare how inosine and uracil can be used in a tRNA anticodon to allow “wobble” when translating an mRNA

Spend no more than 5 minutes writing

A
105
Q

Ribosome synthesises proteins

A
  • Arrows show ribosomes on the Endoplasmic Reticulum making proteins that will be secreted or have roles in membranes
  • A single cell typically contains millions of ribosomes making 10 million peptide bonds per second per cell (in eukaryotes)
106
Q

Which direction is mRNA decoded?

A

From the 5’ to 3’ end and proteins are synthesises starting from their amino-terminus to their carboxyl terminus

107
Q

What does translation occur in?

A

Occurs in ribosomes: complex ribonucleoprotein particles which bind mRNA and tRNA, and contain rRNA and proteins

108
Q

Ribosomes have an Mr of what?

A

over 2.5 million Da

109
Q

In eukaryotes, where do ribosomes assemble?

A

In the nucleolus

110
Q

Tell me the key features of the bacterial E. Coli ribosomal subunit?

A
  • 30S subunit – 16S rRNA + proteins S1- S19
  • Contains the “decoding centre” (DC)
  • Helix 44 of 16S rRNA forms the A and P tRNA binding sites
  • 3’ of 16S rRNA complements the Shine-Dalgarno in mRNA
  • 50S subunit - 23S and 5S rRNAs + proteins L1– L31
  • 23S rRNA forms 6 domains (I-VI)
  • Contains the peptidyl transferase centre (PTC)
  • Contains the peptide exit tunnel
111
Q

What does the ribosome catalyse?

A

Peptide bond formation

112
Q

What was the original thoughts for ribosomes and now what is the final confirmation?

A
  • Originally thought that rRNA was just a scaffold for the proteins
  • However, biochemical and genetic data pointed to a key role for domain V of 23S rRNA in the peptidyl-transferase function
  • Final confirmation that the ribosome is a ribozyme (a ribosome capable of acting like an enzyme) came from the crystal structure of the 50S subunit from the archaebacterium Haloarcula marismortui
  • Ribozyme = an RNA molecule capable of acting as an enzyme
113
Q

Whats the equation for Peptidyl- tRNA to a ‘spent’ tRNA?

A
114
Q

What are two pieces of evidence that ribosomes are ribozymes?

A
  • No ribosomal proteins located in the PTC (peptidyl-transferase centre) – nearest ones are 15-18Å away – too far to take part
  • Ribosomes that are largely depleted of protein still have peptidyl-transferase activity
115
Q

What is each site of the ribosome occupied by?

A

A (occupied by Aminoacyl-tRNA)

P (occupied by Peptidyl-tRNA)

E (occupied by deacylated tRNA, so Exit site)

116
Q

What is required to create a peptide bond?

A

Need the NH of incoming amino acid (attached to tRNA) to attack CO of the growing polypeptide, which is still attached to tRNA

Ends up with extended polypeptide

117
Q

Whats used to break the ester bond between amino acid and tRNA?

A
  • Need to break ester bond between amino acid and tRNA
  • Nucleophilic attack by amino group of the incoming amino acylated tRNA
118
Q
  • The ribosome is a ribozyme
  • Conserved RNA secondary structure around peptidyl transferase center (highlighted bases are masked/disrupted by antibiotics)
  • These are the 3’ A bases from the tRNAs, not the ribosome
  • Crystal structure of a transition state analog revealed several ribosomal (nucleotide) residues that may be involved
  • Only A2486 shown for simplicity
A
119
Q

Mutation of 2’-OH of A76 of peptidyl-tRNA has what effect?

A

Decreases the rate of peptide bond formation by roughly a million-fold

120
Q

Does mutagenesis of rRNA bases in E.Coli e.g. . A2451 (≡A2486), U2506, U2585, A2602, affect peptidyl-transferase reaction?

A

it does not

121
Q

What does ribosome help to position?

A

Ribosome helps to position substrates and water molecules with aids proton transfer and stabilisation of intermediates

122
Q

The ribosome does not work by chemical catalysis but instead how?

A

it instead decreases activation energy needed for the peptide bond formation

123
Q

What does Elongation (EFs/eEFs)…

  • ensure
  • decode
A
  • ensures correct amino acid is added sequentially to the growing protein chain by base pairing of transfer RNA (tRNA) with mRNA
  • decodes 10 – 40 aa per sec with only 1 in 10,000 error rates
  • already seen that accuracy of charging tRNAs with the correct amino acid is key to this, but elongation factors do check fidelity of codon: anticodon pairings
124
Q

During elongation, what does the next aminoacyl-tRNA bind to?

if the codon:anticodon is correct what happens?

Where does peptide bond formation occur?

A

A-site aided by elongation factor EFTu•GTP

If codon:anticodon is correct, EFTu·GTP is hydrolysed to EFTu·GDP

EFTu·GDP is then released and recycled to EFTu·GTP by EFTs

EFTs is a guanine nucleotide-exchange factor (GEF)

EFTu·GDP + GTP –> EFTu·GTP + GDP

Peptide bond -CO-NH- formation occurs in the PTC

125
Q

When translocation occurs what moves and where does it move to?

What does it require?

A

Translocation then occurs peptidyl-tRNA moves to the P-site; the spent tRNA moves to the E-site and exits the ribosome

This requires EFG•GTP which is hydrolysed to EFG•GDP

126
Q

What happens after translocation at the P site?

How much energy does this cost?

A

The next aminoacyl-tRNA binds at A-site; cycle is repeated

“costs” 2x GTP molecules

127
Q

The mechanism of elongation?

A
128
Q

How many steps does translocation occur in?

What happens in these steps?

A

Two steps

  1. the 3’- ends of the tRNAs
  2. Codon: Anticodon pairs
129
Q

What is tRNA always paired with and why?

A

tRNA is always base pairs with mRNA, this prevents frameshifting, ensures accuracy and shows why code is read triplet by triplet

130
Q

Summary of mRNA translation in bacteria

A
131
Q

Another short answer question:

Elongation factors play a crucial role in protein synthesis. Discuss how the three different prokaryotic elongation factors participate in the different steps of the peptide elongation cycle.

A
132
Q

How do Nascent proteins exit through?

A

A “tunnel”

133
Q

What do Nascent chain proteins pass through?

What does it protect and what does this allow?

A

The ribosome exit tunnel

This protects the new chain from inappropriate interactions and this allows it to sample multiple conformations

134
Q

The sequence of amino acids (primary structure) of a protein determines what?

A

It folding and its final shape

135
Q

When does the N-terminal portion start to fold?

A

before the C-terminal region is completed

136
Q

What are many proteins initially synthesised as?

A

Many proteins are initially synthesised as pre- or pro-proteins and are post-translationally modified(phosphorylation, glycosylation etc)

137
Q

What do many proteins bind?

A

Coenzymes (NAD), Cofactors (Zn2+) or prosthetic groups (Haem)

138
Q

Name some eukaryotic elongation factors?

A

eEF1A equivalent to EFTu

eEF1B = EFTs (GEF for recycling eEF1A)

eEF2 = EFG

139
Q

What does phosphorylation of eEF2 reduce?

A

Phosphorylation of eEF2 (in response to rise in AMP, rise in Ca2+) reduces elongation rate

140
Q

How long does the elongation stage continue for?

A

Until a termination codon occupies A-site

141
Q

What happens in protein synthesis in bacteria in the termination stage?

A
  • Ribosome fails to find cognate AA-tRNA
  • Release factors RF1 or RF2 and RF3 bind (RF1/2 mimic tRNA)

- RF1, UAA/UAG

  • RF2, UAA/UGA
  • The final aminoacyl-tRNA ester link is hydrolysed by reaction with H2O and the peptide is released from the ribosome
  • The mRNA and the ribosomal subunits separate from each other
  • RRF recycles the subunits
142
Q

How is eukaryotic termination different to bacterial termination?

A
  • eRF1 for all three stop codons, eRF3 in the GTPase
  • several recycling factors ABCE1 is key one
143
Q

Although prokaryotic and eukaryotic ribosome are similar, most antibiotics bind specifically to bacterial ribosome why?

Explain

A

Because of small structural differences

  • Chloramphenicol - blocks peptidyl transferase
  • Erythromycin - blocks elongation by binding to the 23S rRNA tunnel
  • Tetracycline - prevents amino acyl tRNA binding
144
Q

What do Aminoglycoside antibiotics paromomycin and streptomycin bind to ?

A

Aminoglycoside antibiotics paromomycin and streptomycin bind to decoding centre and induce errors in translation

145
Q

Binding of a correct (cognate) AA-tRNA normally induces what?

What else can induce this change?

A

A conformational change in 30s subunit, to closed state, activating hydrolysis of EFTu•GTP

In the presence of paromomycin, even ‘near-cognate’ AA-tRNAs can induce this conformational change, so that mistakes occur more easily.

146
Q

Whats puromycin?

A

Puromycin - is an analogue of tyrosyl aminoacyl tRNAtyr and causes premature peptide chain termination - also inhibits protein synthesis in eukaryotes

Can use puromycin analogues as tags to assay protein synthesis

147
Q

Overall comparison

A
148
Q

What do we need for protein synthesis?

A
149
Q

Summary

A

Summary

  • Ribosomes are complex molecular machines, with rRNA forming important structural parts e.g. a peptidyl transferase centre
  • PTC facilitates peptide bond formation between two charged tRNAs
  • Translation elongation and termination events in prokaryotes and eukaryotes are similar
  • Charging of tRNAs with correct amino acid ensures high fidelity
  • Fidelity of protein synthesis is also ensured by elongation factors which check mRNA:tRNA codon:anticodon interaction
  • Next lecture: what happens when silent or nonsense mutations occur in the open reading frame?
150
Q

Steps of translation layed out all together as a summary

A
151
Q

Sites of the ribosome?

A

Remember that ribosome has three sites:

  • A (occupied by Aminoacyl-tRNA)
  • P (occupied by Peptidyl-tRNA)
  • E (occupied by deacylated tRNA, so Exit site)
152
Q

Tell me how the bonds of ribosome are created?

A
  • To create a peptide bond, need NH of incoming amino acid (attached to tRNA) to attack CO of the growing polypeptide, which is still attached to tRNA.
  • Ends up with extended polypeptide
153
Q

What elongation factors are used in eukaryotes?

A
  • eEF1A equivalent to EFTu
  • eEF1B = EFTs (GEF for recycling eEF1A)
  • eEF2 = EFG
  • Phosphorylation of eEF2 (in response to rise in AMP, rise in Ca2+) reduces elongation rate
154
Q

Tell me about how translation is terminated in eukaryotes?

A
  • Elongation continues until a termination codon occupies A-site
  • Ribosome fails to find cognate AA-tRNA
  • Release factors eRF1 and eRF3 bind (eRF1 mimics tRNA)
  • The final aminoacyl-tRNA ester link is hydrolysed by reaction with H2O and the peptide is released from the ribosome
  • The mRNA and the ribosomal subunits separate from each other
  • ABCE1 recycles the subunits
155
Q

Nonsense- mediated decay (NMD) is a type of mRNA surveillance, what is this?

A
  • A surveillance mechanism to detect and destroy aberrant mRNAs containing Premature Termination Codons (PTCs) BEFORE translation
  • Prevents accumulation of proteins with C-terminal truncations, which could create inactive or even dominant negative versions
156
Q

Tell me about splicing and the exon junction complex (EJC)

A
  • Protein complex known as the exon junction complex (EJC) is deposited during splicing.
  • It is placed 20-24 nucleotides upstream of each splice site.
  • EJC contains various proteins, most important is UPF3
  • During export UPF2 binds to UPF3 in EJC
157
Q

Tell me about translation of normal mRNA

A
158
Q

Tell me about translation of mRNA containing PTC (nonsense mRNA)

What is nonsense mRNA?

A
159
Q

Tell me about the fate of mRNAs containing a PTC?

what the SURF complex

A
160
Q

Tell me some diseases associated with the presence of PTCs?

A
  • Duchenne muscular dystrophy (DMD)
  • Familial adenomatous polyposis
  • Hereditary breast and ovarian cancer
  • Polycystic kidney disease
  • Cystic Fibrosis

PTCs may account for 90% of identified mutations in these diseases!

161
Q

Ribosomes can read through the presence of PTC in the presence of what?

A

Gentamicin

162
Q

Is therapeutic intervention possible with similar aminoglycosides?

A

PTC124 (Translaren/Ataluren) is available for DMD treatment in the UK, but CF trials failed, even though they showed promise in mice with G542X mutation of the CFTR

163
Q

No stop codon?

A
164
Q

Mutations or mRNA features that stall the ribosome?

A
165
Q

In humans Leu is 6x more likely to be coded by CUG than a UUA, why is this?

A
  • Useful to stop ribosomes accelerating too quickly at start of ORF
  • Help to give enough time for the protein to fold
  • May be helpful in regulating protein expression
  • e.g. in ZEB2 translation of Leu(UUA)-Gly(GGU)-Val(GUA) causes ribosomal pausing and limits protein production but changing the codons to the common ones does not
  • BUT pausing can cause no go decay
166
Q

summary of lecture 18

A
  • Comparing the three surveillance mechanisms, all can lead to destruction of an aberrant mRNA
  • This is an important quality control mechanism
  • e.g., with No-Go decay, if the collided ribosomes do not dissociate, the reading frame can shift to the +1 frame and then make an aberrant protein
167
Q

Explain the 48S pre-initiation complex (PIC) formation and mRNA circularisation and the similaries to prokaryotes and the key differences

A
168
Q

Diagram explaining control of eLF2 availability

A
169
Q

What are the steps to the function and control of eLF2?

A
170
Q

What does the activation of PKR in response to viral RNA cause?

A

This causes a reduction in protein synthesis of both host and viral mRNA, which should prevent further infection

171
Q

inhibition of PKR by viral proteins

A
172
Q

Control of eLF4E availability

Include about the role of 4E-BPs

A
173
Q

Cleavage of eLF4G is done by what?

A
174
Q

The cleavage of eLF4G and polysome collapse following what?

A

poliovirus infection

175
Q

Tell me about some translational control- mRNA specific mechanisms

A

Secondary and tertiary structures formed by base-pairing of single stranded RNA – depends on length and G-C content

  • Internal Ribosome Entry Sites (IRESs)
    • May need non-canonical factors (ITAFs)
176
Q

Tell me about internal ribosome entry and when it was first discovered?

A

First discovered in picornaviruses, used to maintain synthesis of viral proteins at expense of host cell cap-dependent translation (Jackson 2005)

177
Q

Information about translation and SARS-COV2

A
  • Cellular mRNAs are degraded following infection, so there are less host cell messages to translate
  • This appears to be dependent on the viral non-structural protein 1 (Nsp1)
  • Even those transcripts which are produced in response to the infection are not translated
  • The Nsp1 blocks the mRNA channel of the 40S subunit
  • General protein synthesis is inhibited, including translation of genes of the innate immune response (e.g., IFN-β)
  • 10 of the 27 viral proteins bind to specific host RNAs (e.g., eEFs)
  • Nsp16 disrupts splicing
  • Nsp8 and Nsp9 stop N-terminal signal peptide of secreted/membrane proteins from being recognised
178
Q

Summary of lecture 19

A
  • mRNA surveillance acts as a final quality control step to ensure only a minimal amount of protein is translated from aberrant mRNAs
  • All mechanisms result in mRNA decay.
  • Nonsense mediated
  • Non-stop
  • No go
  • Viruses ensure their own protein synthesis by many mechanisms, which is one of the reasons it is so difficult to develop effective antiviral strategies
  • Many disrupt the host protein synthetic machinery directly, hijacking the translation to ensure viral proteins are made

BIOL2010 (7 decks)

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