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Immunology > Lecture 20, 21 > Flashcards

Lecture 20, 21 Flashcards

1
Q

What happens in translation

A
  • Protein synthesis

- Nucleotide sequence in mRNA is translated by the tRNAs into an amino acid sequence in a polypeptide

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2
Q

What is one reasons why understanding the mechanism of protein translations important

A

improved protein expression systems in biotechnology

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3
Q

How does prokaryotes and eukaryotes differ in terms of where translation takes place

A

• in prokaryotes, protein synthesis can occur simultaneously with
transcription
• in eukaryotes DNA is confined to the nucleus; RNA is made in the
nucleus (transcription) but exported to the cytoplasm; protein
is synthesized in the cytoplasm

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4
Q

protein synthesis is usually proportional to what? Give an example

A
  • the amount of RNA
    in a cell
    e.g. pancreatic cell - RNA-rich, produces large amount of proteolytic enzymes for digestion
    e.g. muscle cell - RNA-poor, very low lvl of protein synthesis
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5
Q

What are the 3 important RNA players in translation?

A

mRNA, tRNA, rRNA

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6
Q

Role of mRNA in translation?

A

the intermediary
between gene and protein – provides the message that is read” by the tRNA adaptors and
translated into an amino acid sequence

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7
Q

Role of tRNA in translation?

A

the key or adaptor –

reads the genetic code, brings amino acids to the growing polypeptide chain

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8
Q

Role of rRNA in translation?

A

in ribosome, provides a
scaffold for protein synthesis, catalyzes peptide bond
formation

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9
Q

How does the enzyme tRNA synthetase start translation?

A

It attaches an AA onto one end of a tRNA. Next, the tRNA brings that aa to the growing polypeptide in the ribosome, selected by base pairing between a three-nucleotide anticodon on the tRNA and the three-nucleotide codon on mRNA.

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10
Q

What dictates which tRNA-aa complex will bind?

A

The nucleotide sequence of the mRNA dictates which tRNA-aa complex will bind.

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11
Q

What catalyzes the transfer of the amino acid from the tRNA to the growing polypeptide chain.

A

The ribosome

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12
Q

How does a tRNA synthetase know which AA to add to which tRNA?

A

Each tRNA is specific for a given AA and each tRNA synthetase is specific for a given tRNA and for its cognate AA. The synthetase recognizes the 3D structure of the tRNA,
including the anticodon and other parts of the tRNA – large interaction surface.

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13
Q

What is tRNA’s 2D representation

A

Cloverleaf because of base pairing

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14
Q

How many tRNA for each AA

A

At least one

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15
Q

What AA’s are at the 5’ and 3’ end of tRNA

A

most tRNAs have a G at the 5’ end and CCA at 3’ end

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16
Q

tRNA has how many arms/loops?

A

4

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17
Q

What part of the tRNA attaches to the carboyl end of an AA?

A

3’ end of the amino acid arm

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18
Q

The anticodon loop base pairs with what

A

codon on mRNA

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19
Q

Purpose of D loop and T-psi-C loop?

A

the D loop and the TyC loop (T-psi-C arm, T loop, T arm) contain modified
nucleotides (e.g., y, pseudo-uridine) and contribute to the 3D structure of the tRNA

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20
Q

What modifications are done to tRNA after transcription? Purpose?

A
  • some of the tRNA bases are modified after transcription (processing)
  • modifications may stabilize 3D structure, help recognition by tRNA synthetase
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21
Q

In what direction are the tRNA and mRNA strands aligned?

A
  • They are antiparallel
  • mRNA is 5’->3’ so tRNA is 3’->5’
  • 1st base in codon of mRNA pairs with 3rd base in anticodon on tRNA
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22
Q

What is degeneracy?

A

in the genetic code: there are 64 codons for 20 amino acids so some amino acids are encoded for by more than one codon.
- A given tRNA will only bind to its cognate (i.e., matching) AA, but some tRNAs can base pair with more than one codon, and some amino acids have more than
one tRNA.

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23
Q

During protein synthesis, the sequence of nucleotides in the mRNA is read by each tRNA from the 5’ to 3’ end in sequential sets of?

A

Three nucleotides (codons)

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24
Q

How many possible reading frames are there?

A
  • 3

- 1st reading frame is correct, 2nd reading frame is one shift to the right, 3rd is 2 shifts to the right

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25
Q

What is a frameshift mutation

A

insertion or deletion of one or two bases will shift the reading frame so that all AA encoded from that point on will be diff, resulting in a diff protein

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26
Q

What happens when a frameshift results in a stop codon

A

terminates protein synthesis, resulting in a truncated protein that will likely not fold properly and will be degraded

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27
Q

If mRNA: 5’-AUGGCUUAUGACCGCAUGAUC-3’

what is DNA coding and template strand?

A

5’ ATGGCTTATGACCGCATGATC-3’ – coding strand

3’-TACCGAATACTGGCGTACTAG-5’ – template strand

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28
Q

What is the wobble position?

A
  • 3rd position

- can tolerate a mismatch or non-WC base-pairing.

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29
Q

anticodons in some tRNAs contain the nucleotide inosate (I) at the first position of the anticodon (5’ end), which pairs with the third base of the codon. Inosate contains the base hypoxanthine, which can H-bond with which nucleotides?

A

A, U, and C

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30
Q

How is hypoxanthine made?

A

deamination of the adenine or guanine base at position 1 of the tRNA anticodon

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31
Q

Why in anticodon allows for stabilization of wobble pairs?

A

The flexibility and environment of the

anticodon both allow and stabilize wobble pairs.

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32
Q

Each AA has how many different tRNA synthetase?

A

One

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33
Q

What are the two steps in loading of the tRNA? Both reactions occur on and are catalyzed by what?

A
  1. The AA is first activated by forming a high-energy linkage to the a-phosphate of ATP - one high-energy bond (~) is broken (ATP -> AMP + PPi) and another is formed (AMP~AA) (Delta G approximately = 0)
  2. The AMP-linked carboxyl group on the AA is transferred to the 2’ or 3’ hydroxyl group of A at the 3’ end of the tRNA. This is also a high-energy linkage.
    - tRNA synthetase
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34
Q

Where does ATP and AA fit in tRNA synthetase?

A

both ATP and aa fits

into deep cleft in tRNA synthetase’s active site pocket

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35
Q

Two methods of discrimination by the tRNA synthetase?

A
  1. the correct AA has the highest affinity for the active site pocket of the
    synthetase (larger aa will be excluded);
  2. when tRNA forms a bond with an aa, the bound aa is pushed into a second editing site pocket. Correct aa are rejected at the editing site so they remain bound to the tRNA; incorrect aa are hydrolysed from the tRNA at the editing site and released.
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36
Q

How are ribonucleoproteins made

A

RNA and protein ribosome components can be purified separately and reconstituted

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37
Q

Reconstituted ribosomes are active, showing that

A
  • all ‘essential’ components of ribosomes are currently known
  • ribosomes have capacity for self-assembly
  • mixing and matching components of ribosomes (from different organisms) is possible
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38
Q

Fxn of large subunit?

A

Catalyzes peptide bond formation

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39
Q

Fxn of small subunit?

A

Provides framework on which tRNA can be accurately matched to the codon of mRNA

40
Q

Where do the two ribosomal subunits assemble?

A

On mRNA

41
Q

What are the numbers for prokaryotic ribosome and eukaryotic ribosome (and the # for subunits to make them)

A

Prokaryotic - 50S + 30S = 70S

Eukaryotic - 60S + 40S = 80S

42
Q

RNA in the ribosome is responsible for what?

A

Rhe ribosome structure, for positioning itself on the mRNA, and for catalytic activity.

43
Q

What are the 3 binding sites for tRNAs? Where are they located

A
  • A, P, and E site

- P and A site spans both large and small subunit while E spans mostly just large subunit

44
Q

Where does the Shine-Dalgarno sequence bind?

A

3’ end of 16S RNA

45
Q

At which terminus does the nascent polypeptide chain emerge first after translation?

A

N-terminus emerges first

46
Q

What is the Shine-Dalgarno part of?

A

16S rRNA

47
Q

What do the A, P, and E sites represent?

A
  • A-site is for aminoacyl-tRNA
  • P-site is for peptidyl-tRNA
  • E-site is for exit
48
Q

How many binding sites for RNA do ribosomes have

A

4

49
Q

Where does the newly-synthesized polypeptide exit?

A

through a hydrophobic tunnel in the 50S subunit at the

site of peptide bond formation.

50
Q

What can fit in the hydrophobic tunnel in the 50S subunit?

A

~30 AA, may be room for a-helix (form secondary structures right in helix)

51
Q

Where does the peptide chain remain throughout translation?

A

P-site, it is passed from one tRNA to the next

52
Q

Is Met only found at first nuc of polypeptide?

A

No, all organisms have two tRNAs that recognize AUG - one
codes for Met in internal positions in the protein and the
other is for the N-terminal Met – the initiation site on the
ribosomes recognizes the unique initiating tRNA-Met

53
Q

In bacteria, what is the N-terminal Met incorporated at the AUG start codon called? And the corresponding tRNA?

A
  • N-formylmethionine or fMet

- tRNA^fMet

54
Q

How is Met turned into fMet for bacteria?

A

the Met-tRNA synthetase adds Met to tRNAfMet and the
bound Met is then formylated by a transformylase that
recognizes tRNAfMet

55
Q

What accepts tRNA^fMet?

A

Special ribosome initiation site

56
Q

How do prokaryotic’s tRNA^fMet and eukaryotic Met differ in terms of position of insertion?

A
  • fMet cannot be inserted into a growing polypeptide chain anywhere but at the 1st position
  • Met is used at all AUG sites regardless of their position in the protein sequence
57
Q

In eukaryotes how is the initiating tRNA^Met diff from tRNA^Met

A

a special initiating
tRNAMet is still used for the N-terminal Met, which is distinct
from the tRNAMet used at internal positions (the Met is the same everywhere but the initiating tRNAMet is distinct from that of the “regular” tRNAMet, which adds Met everywhere but at the
start codon)

58
Q

What is needed to make fMet-tRNA^fMet in bacteria?

A
  • Met + tRNA^fMet
  • Met-tRNA synthetase
  • Transformylase
59
Q

What is needed to make Met-tRNA^Met in bacteria?

A
  • Met + tRNA^Met

- Met-tRNA synthetase

60
Q

What is needed to make Met-tRNA^Met in eukaryotes?

A
  • Met & tRNA^Met

- Met-tRNA synthetase

61
Q

What does polycistronic mean?

A

More than one protein encoded on the mRNA

62
Q

What does initiation of protein synthesis in bacteria require?

A
  • the 30S ribosomal subunit (small subunit)
  • the mRNA coding for the polypeptide to be synthesized
  • the initiating fMet-tRNAfMet
  • a set of three protein initiation factors - IF-1, IF-2 and IF-3
  • the 50S ribosomal subunit (large subunit)
  • Mg2+
  • GTP
63
Q

There are often several AUG codons in a message, coding for Met in the protein. How does the initiation complex know at which AUG to start synthesis?

A

an initiation signal called the Shine-Dalgarno sequence 8-13 bp to the 5’ side of the AUG initiation codon – this sequence base pairs with a complementary sequence on the 16S RNA of the small subunit and precisely
positions it for initiation of translation.

64
Q

How many Shine-Dalgarno sequences are there for polycistronic mRNA? Allowing what?

A

Polycistronic mRNA has a Shine-Dalgarno sequence for each cistron (protein coding region), allowing
each protein to be translated independently and at
different levels.

65
Q

Describe initiation of protein synthesis in bacteria with initiation factors

A
  1. 30S subunit binds to IF-1 and IF-3, then the mRNA
  2. IF-2-GTP binds the 30S subunit and recruits fMet-tRNA^fMet which base pairs with start codon
  3. The 50S subunit associates, IF-2 hydrolyzes GTP, and IF-1 & IF-2 & IF3 dissociate leaving the initiation complex
66
Q

Function of IF-1, IF-2, IF-3 (prob don’t need to know)

A
  • IF-1 binds to the A site and prevents addition of new tRNAs during initiation
  • IF-3 preents premature association with 50S subunit
  • IF-2-GTP binds the 30S subunit and recruits fMet-tRNA^fMet which base pairs with start codon
67
Q

What is the initiation complex in bacteria?

A

70S complex

68
Q

What is the first elongation step in bacteria?

A

Binding of the second amino-acyl-tRNA

69
Q

What does elongation require

A

70S initiation complex, aminoacyl tRNAs, and a set of three Elongation Factors, EF-Tu (think EF-2, like IF-2), EF-Ts and EF-G, and GTP

70
Q

What happens in the first step of elongation in bacteria?

A
  • the appropriate aminoacyl-tRNA binds to a complex of GTP-bound EF-Tu (read EF-2, analogous to fMet-tRNAfMet binding to IF-2 during initiation)
  • this new complex binds to the A-site of the 70S initiation complex (remember, fMet-tRNAfMet is in the P site)
  • GTP is hydrolyzed and EF-Tu-GDP complex is released
  • the GTP-EF-Tu complex is regenerated with the help of EF-Ts
71
Q

for initiation and elongation in both
prokaryotes and eukaryotes, the aminoacyltRNAs
must form a complex with a _____

A 
a GTP-bound
protein factor (IF-2; eIF-2; EF-Tu, eEF1a) to bind to the ribosome
72
Q

Where is the peptide bond formed during translation

A

between the carboxyl group at the end of the growing polypeptide chain and the free amino group on the incoming AA (nucleophilic attack of the electron deficient carboxyl carbon by the amino nitrogen of the incoming AA).

73
Q

Is the formation of each peptide bond during translation is energetically favourable or not?

A

Yes because of the high energy linkage of the carboxyl end of the peptide chain to the tRNA molecule

74
Q

What is the peptidyltransferase rxn catalyzed by?

A

the 23S rRNA (ribozyme) in the 50S subunit

75
Q

What is the second elongation step in bacteria?

A

Formation of the first peptide bond

76
Q

What occurs in the first peptidyltransferase?

A

fMet is transferred from tRNAfMet to the second amino acid, AA2, which is attached to its tRNA in the A site. As
part of this process the tRNA of AA2 shifts its aminoacyl arm (i.e., the 5’ and 3’ ends) into the P site of the large (50S) subunit, leaving the rest of the tRNA in the A site of the small (30S subunit). Similarly, the aminoacyl arm of the “deacylated” or
“uncharged” tRNAfMet shifts from the P site to the E site. Thus, the peptide chain remains on the P-site of the 50S subunit throughout translation – is passed from one tRNA to the next

77
Q

What is the 3rd step in elongation in bacteria?

A

Translocation

78
Q

What occurs during the 3rd step of elongation in bacteria, aka what is translocation?

A
  • Ribosome undergoes conformational shift to move along the mRNA one codon in the 5’-3’ direction so the deacylated tRNAfMet and 1st AUG codon now fully in the
    E-site, the peptidyl-tRNA and 2nd codon are in the P-site, and a third vacant codon is in the A-site, ready to accept a new aminoacyl-tRNA. This shift is called translocation
  • The deacylated tRNA^fMet dissociates from the E-site.
79
Q

What does translocation require?

A

requires a translocase, EF-G, and energy from

GTP hydrolysis

80
Q

Fxn of EF-G during translocation?

A

EF-G mimics the structure of the EF-Tu-tRNA complex, which may allow it to bind to the A-site and displace the
peptidyl-tRNA

81
Q

What is the purpose of GTP hydrolysis during translocation?

A

GTP hydrolysis releases EF-G from the A site, opening up
the A site for another aa-tRNA-EF-Tu to dock, based on base
pair complementarity with the codon at the A site.

82
Q

When does elongation stop?

A

Until a stop codon is reached

83
Q

The polypeptide remains attached to the tRNA of the ___

A

most recently-inserted aa.

84
Q

Do tRNAs bind to stop codons?

A

No because stop codons do not code for any AA

85
Q

What happens in bacteria when a stop codon occupies the A-site?

A

a termination/release factor (RF-1 or -2, depending on the

stop codon) binds to the A site.

86
Q

What are RFs (release factors)

A

Proteins that mimic the tRNA structure and can bind in the A-site when a stop
codon is present.

87
Q

What does RF binding result in (3)?

A

• hydrolysis of the terminal peptidyl-tRNA bond (the growing
polypeptide transfers from tRNA to a water molecule rather than a new aa)
• release of the last uncharged tRNA and the polypeptide
• dissociation of the 70S ribosome into the 30S and 50S subunits

88
Q

What kind of post-transtionally modifications are there for bacteria

A
  • the formyl group, the N-terminal fMet, and often additional N-terminal or C-terminal residues may be enzymatically removed.
  • Disulfide bonds may form
89
Q

What kind of post-transtionally modifications are there for eukaryotes

A
  • 50% of eukaryotic proteins are N-acetylated
  • N-terminal “signal sequences” are removed for secreted
    proteins, and some proteins undergo further proteolytic processing.
  • Individual AA may be covalently modified by addn of phosphate, carbohydrate, isoprenyl, prosthetic
    groups
90
Q

When are the secondary and tertiary structure of a protein made?

A

Acquired as it emerges from a ribosome, starting from the N-terminal end. Some proteins require the help of chaperones to fold properly. Many proteins will associate
with other proteins in their final active form.

91
Q

What happens to failed translation products?

A

Degraded by proteasome

92
Q

What is proteasome

A
  • A protease machine
  • cooperate with other factors to regulate
    the timely degradation of native proteins
93
Q

Why is a chaperone required for some proteins?

A
  • To shield hydrophobic surfaces that are exposed b4 the protein has reached its native conformation
  • Prevent proteins from aggregating via exposed hydrophobic regions, allowing them to reach their folded state
  • Help misfolded proteins re-fold correctly
94
Q

If misfolded proteins are not refolded correctly what happens

A

rapidly destroyed by a complex ATP-dependant protease complex called a proteasome

95
Q

What happens to proteins with exposed hydrophobic regions?

A

marked for destruction by covalent binding of multiple copies of a small protein called ubiquitin, which allows them to be recognized by the proteasome. Ubiquitinated proteins
are bound by the proteosome, unfolded and degraded into short peptides.

Immunology (14 decks)

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