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bioc 299 > Translation > Flashcards

Translation Flashcards

1
Q

How did Crick suggest mRNA is read?

A

by hydrogen bonding

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2
Q

According to Crick, how many tRNAs are required to read mRNA sequences? why?

A

a minimum of 20 mRNAs because there are 20 amino acids and Crick proposed that

amino acid is carried to mRNA by a tRNA molecule that can read the mRNA sequence = each amino acid needs a tRNA

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3
Q

What did Gamow propose the mRNA is read as?

A

‘words’ but really codons

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4
Q

What does protein biosynthesis require?

A

ribosomes, mRNA, tRNA, and protein factors

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5
Q

Where are proteins synthesized?

A

the ribosome

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6
Q

What ensures the right order of amino acids for the synthesis of the polypeptide chain?

A

mRNA and tRNA which are bound to the ribosome

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7
Q

What is a codon?

A

a sequence of 3 bases that specifies one amino acid

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8
Q

What is the genetic code?

A

the translation for the language of DNA (nitrogenous bases) into the language of proteins (amino acids)

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9
Q

How many possible codons are there for the 20 amino acids?

A

4^3 = 64 codons

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10
Q

Why are there 64 codons?

A

because there’s 4 base pairs (A, G, C, T) in DNA and codons are groups of 3 bases = 4^3 = 64

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11
Q

How did they discover that codons must be read as groups of 3?

A

because if they were 2 bases long, 4^2 = 16, which isn’t enough for the 20 amino acids

so it must be 4^3 = 64

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12
Q

What are the 6 qualities of the genetic code/codons?

A

unambiguous
directional
nonoverlapping
commaless
degenerate
universal

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13
Q

What does it mean that the genetic code is nonoverlapping?

A

no bases are shared between consecutive codons = the ribosome reads 3 bases at a time

ex. GACCUA is read by the ribosome as GAC then CUA, not GAC then ACC then CCU then CUA

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14
Q

What does it mean that the genetic code is commaless or not punctuated?

A

there’s no bases that serve as punctuations between the codons that are read by the ribosome - all the bases contribute to the codons

ex.
punctuated: AUGCCAUCGCA = Met comma His comma Ala -> this would actually give different amino acids because it would be read as AUG CCA UCG CA_

commaless: AUGCAUGCAU = Met His Ala

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15
Q

What does it mean that the genetic code is degenerate?

A

it’s redundant

there’s 64 codons but only 20 amino acids so some amino acids have multiple codons

ex. UUU and UUC both code for Phe

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16
Q

What does it mean that the genetic code is unambiguous?

A

one codon = one amino acid

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17
Q

What does it mean that the genetic code is universal?

A

the same codons code for the same amino acids in all organisms

the only difference is in human mitochondria, mitochondria have different stop codons

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18
Q

What is different about the standard genetic code and the human mitochondrial genetic code?

A

different stop codons used

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19
Q

What is a reading frame?

A

the framework for how the genetic code is read = the starting point for the ribosome to read the sequence in groups of 3 nucleotides

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20
Q

What were the 3 key experiments that deciphered the genetic code?

A
  1. Nirenberg & Matthaei: found the first 3 codons (UUU = Phe, CCC = Pro, AAA = Lys) using protein synthesizing extract and RNA template
  2. Nirenberg & Ochoa: tested all 64 codons individually and found that 50 worked clearly to determine which tRNAs bind to RNA
  3. Kohrana: figured out the last 14 codons by synthesizing polyribonucleotides with defined sequences and protein synthesizing extract
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21
Q

What synthesizes proteins?

A

ribosomes

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22
Q

In what direction of the RNA strand do ribosomes synthesize proteins?

A

from the N-terminus to the C-terminus

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23
Q

What does translation require to begin?

A

activated/charged aminoacyl-tRNAs

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24
Q

What are the 3 steps of translation?

A

initiation, elongation, termination

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25
Q

T or F: translation does not require energy input

A

false, it needs energy input

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26
Q

What shape does the secondary structure of tRNA have?

A

cloverleaf

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27
Q

Describe the secondary structure of tRNA

A

the 3’ end always has CCA-OH and it is where the amino acid is attached

there is an anticodon loop which basepairs with mRNA template in an antiparallel fashion

the 5’ end is always phosphorylated

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28
Q

Approximately how many nitrogenous bases in tRNA are modified (ie., not A, C, G, or U)?

A

~25%

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29
Q

Who discovered the first base sequence of tRNA?

A

Robert Holley

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30
Q

Which part of the tRNA is the acceptor stem?

A

the phosphorylated 5’ terminus

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31
Q

Which part of the secondary structure of tRNA is the anticodon arm?

A

the anticodon loop

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32
Q

What kind of base pairing occurs in the tRNA?

A

Hydrogen bonds between base pairs, base pairing follows non-Watson and Crick pairing (NON-CANONICAL)

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33
Q

T or F: base pairing in tRNA is non-canonical

A

true

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34
Q

Describe the tertiary structure of tRNA

A

L shaped

non-canonical base pairing with hydrogen bonds
Amino acid at 3’ end

Codon attached in antiparallel fashion to the anticodon arm

arms are stacked and there’s 2 segments of double helix

compact

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35
Q

What are the “arms” or “loops” of the secondary structure of tRNA?

A

T (modified base) C arm
D arm
variable arm

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36
Q

What are the possible modified nucleotides?

A

inosine
5-methylcytidine (mC)
dihydrouridine (UH2)

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37
Q

What is beneficial about the structure of the tertiary structure of tRNA?

A

the anticodon loop is easier to observe

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38
Q

What do the anticodons in the anticodon loop/arm of tRNA base pair with?

A

the codons of mRNA

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39
Q

What direction do the codons of mRNA run and what direction do the anticodons of tRNA run?

A

they run antiparallel

ex.
mRNA codons: 5’- G C C -3’
tRNA anticodons: 3’- C G G - 5’

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40
Q

What results from the bending of the anticodon loop of tRNA?

A

a “wobble” base in the 3rd position of the mRNA codon / 1st position of the tRNA anticodon

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41
Q

Which of the bases is the wobble base if:

mRNA: 5’ - G C C - 3’
tRNA: 3’ - C G G - 5’

A

the wobble position is the 3rd base in the tRNA anticodon

in this example it’s the second G (G closest to 5’)

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42
Q

What does the wobble position / wobble pairing mean?

A

the first 2 positions in the mRNA codon are canonically (Watson-Crick) base paired with the tRNA anticodon

the 3rd position (closest to the 3’ end) of the mRNA is the wobble position and can allow non-canonical (non-Watson-Crick) base pairing

this means that the tRNA base that pairs with the nucleotide at the wobble position can be something different than in canonical base pairing

43
Q

What function does the wobble position serve?

A

it allows there to be less tRNAs than codons because one tRNA can recognize and basepair with multiple codons

44
Q

What base(s) at the 3’ end of the mRNA codon (wobble position) can base pair with the following base at the 5’ end of the tRNA anticodon?

base at 5’ end of tRNA anticodon = I* (Modified base)

A

if the base at the 5’ end of the tRNA anticodon is a modified base, the tRNA can pair with an mRNA codon that has either A, C, or U bases at the 3’ position

45
Q

What base(s) at the 3’ end of the mRNA codon (wobble position) can base pair with the following base at the 5’ end of the tRNA anticodon?

base at 5’ end of tRNA anticodon = G

A

if the base at the 5’ end of tRNA anticodon is G, the tRNA can pair with an mRNA codon that has C or U at the 3’ end

46
Q

What base(s) at the 3’ end of the mRNA codon (wobble position) can base pair with the following base at the 5’ end of the tRNA anticodon?

base at 5’ end of tRNA anticodon = U

A

if the base at the 5’ end of the tRNA anticodon is a U, the tRNA can pair with an mRNA codon that has A or G at the 3’ end

47
Q

What base(s) at the 3’ end of the mRNA codon (wobble position) can base pair with the following base at the 5’ end of the tRNA anticodon?

base at 5’ end of tRNA anticodon = A

A

if the base at the 5’ end of the tRNA anticodon is A, the tRNA can pair with an mRNA codon that has U at the 3’ end

this is normal canonical base pairing = no wobble

48
Q

What base(s) at the 3’ end of the mRNA codon (wobble position) can base pair with the following base at the 5’ end of the tRNA anticodon?

base at 5’ end of tRNA anticodon = C

A

if the base at the 5’ end of the tRNA anticodon is C, the tRNA can only pair to an mRNA codon with G at the 3’ end

this is normal canonical base pairing = no wobble

49
Q

Which 3 bases at the 5’ end of the tRNA anticodon have the wobble effect and can be paired non-canonically?

A

anticodon I* (modified) = codon A, C, or U
anticodon G = codon C or U
anticodon U = codon A or G

50
Q

Which 2 bases at the 5’ end of the tRNA anticodon do not have the wobble effect and can only be paired canonically?

A

anticodon A = codon U
anticodon C = codon G

51
Q

With the following mRNA codon: 5’ - G U A - 3’

What is the corresponding tRNA anticodon?

A

codon:
5’ - G U A - 3’

anticodon:
3’ - U A C - 5’
or
3’ - U A U - 5’

52
Q

With the following tRNA anticodon: 3’ - C G I -5’

What is the corresponding mRNA codon?

A

anticodon:
3’ - C G I - 5’

codons:
5’ - G C A - 3’
5’ - G C U - 3’
5’ - G C C - 3’

53
Q

What needs to happen before translation is initiated?

A

an amino acid needs to be activated and an aminoacyl-tRNA needs to form

54
Q

How many steps does the reaction that charges the amino acid and forms the aminoacyl-tRNA have?

A

2:

one that activates the amino acid
AA + ATP –> Aminoacyl-AMP + PPi

one that forms the aminoacyl-tRNA
aminoacyl-AMP + tRNA –> Aminoacyl-tRNA + AMP

55
Q

Which enzyme catalyzes the two steps of the reaction that activates the amino acid and forms the aminoacyl-tRNA?

A

aminoacyl-tRNA synthetase

56
Q

What is the overall reaction for the formation of aminoacyl-tRNA?

A

amino acid + ATP + tRNA –> aminoacyl-tRNA + AMP + PPi

57
Q

Does the reaction that forms aminoacyl-tRNA require an input of energy?

A

yes, ATP is required to activate the amino acid before it’s transferred to tRNA

58
Q

How many different aminoacyl-tRNA synthetases are there?

A

20, one for each of the amino acids

59
Q

T or F: aminoacyl-tRNA synthetases are extremely specific

A

true = there is one for each amino acid because joining the correct amino acid to the correct tRNA is essential

60
Q

How are amino acids selected?

A

charge, size, hydrophobicity

61
Q

How are tRNAs selected?

A

their unique structural features (acceptor arm and anticodon)

62
Q

What happens if the wrong amino acid is joined to the wrong tRNA?

A

the amino acid will be incorporated during translation and could change the end protein product

63
Q

How big is the bacterial ribosome?

A

70S

64
Q

How big is the eukaryotic ribosome?

A

80S

65
Q

What is the unit used to measure the size, shape and density of the ribosomes?

A

Svedberg (S)

66
Q

How many subunits does a bacterial ribosome have? what are their measurements?

A

2 subunits

large: 50S (contains 5S and 23S rRNA)
small: 30S (contains 16S rRNA)

67
Q

What are the measurements for the large and small subunits of the bacterial ribosome?

A

large is 50S with 5S rRNA and 23S rRNA

small is 30S with 16S rRNA

68
Q

How many subunits does a eukaryotic ribosome have?

A

2 subunits

large: 60S with 5S, 28S, and 5.8S rRNA
small: 40S with 18S rRNA

69
Q

What are the measurements for the large and small subunits of the eukaryotic ribosome?

A

large: 60S with 5S, 28S, and 5.8S rRNA

small: 40S with 18S rRNA

70
Q

What is used to initiate translation in bacteria? Eukaryotes?

A

Both use a tRNA^met

bacteria use N-formylmethionyl-tRNAf (tRNA^fmet)

eukaryotes use methionyl-tRNAi (tRNA^met)

71
Q

What direction does the synthesis of a polypeptide chain run?

A

N-terminus to C-terminus

72
Q

What is the initial N-terminal amino acid of all proteins in prokaryotes? which tRNA is used to form the aminoacyl-tRNA?

A

N-formylmethionine (fmet) binds with N-formylmethionine-tRNAf

73
Q

What is the start signal at the beginning of the mRNA sequence for translation to begin in prokaryotes and eukaryotes?

A

both tRNAfmet and tRNAmet have an anticodon 3’-UAC-5’ that base pairs to the mRNA codon 5’-AUG-3’

AUG is the start signal codon

AUG codes for Methionine

74
Q

What does the start signal mRNA codon code for?

A

methionine

75
Q

What are initiation factors? What are they in prokaryotes? eukaryotes?

A

proteins that help form the initiation complex

prokaryotes: IF-1, IF-2, IF-3
eukaryotes: eIFs (8+)

76
Q

What is the translation initiation complex composed of? When is it assembled?

A

ribosomal subunits
mRNA template
initiator tRNA molecule
protein initiation factors

assembled at the beginning of mRNA coding sequence

77
Q

Describe the steps of the formation of the initiation/translation complex in prokaryotes

A
  1. the 30S subunit binds with IF3 and IF1
  2. GTP is added, N-formylmethionine-tRNA (fmet-tRNAfmet) binds with IF2 and the mRNA transcript at the Shine-Dalgarno sequence and aligns the AUG start codon to the UAC anticodon
  3. 30S subunit binds with mRNA, fmet-tRNA (bound to IF2) anticodon UAC pairs with AUG start codon
  4. IF2 is released
  5. 50S subunit joins, GDP + PPi + IF1 + IF2 released

final: 70S initiation complex is formed which includes:
- 50S ribosomal subunit
- 30S ribosomal subunit
- mRNA transcript with AUG start codon aligned and paired with fmet-tRNAfmet UAC anticodon

78
Q

T or F: energy input is required for the formation of the translation initiation complex

A

true, GTP is needed to bind the fmet-tRNAfmet*IF2 with the 30S subunit and the mRNA transcript

79
Q

What is the Shine-Dalgarno sequence?

A

it’s a segment of mRNA that is upstream (closer to 5’) from the AUG start sequence

it’s purine-rich

5’-GGAGGU-3’

it functions as the ribosomal binding site in prokaryotic translation

80
Q

What is the function of the Shine-Dalgarno sequence?

A

only in prokaryotes

it’s the binding site for the 30S ribosomal subunit to allow for the correct alignment of the AUG start codon with the UAC anticodon

81
Q

Why is it essential for the 50S subunit of the 70S prokaryotic ribosomal subunit to bind and be part of the translation complex?

A

the 50S ribosomal subunit has the 3 binding sites (EPA) for tRNA to allow for elongation

82
Q

What are the 3 tRNA binding sites on the 50S ribosomal subunit?

A

A: aminoacyl site
P: peptidyl site
E: exit site

83
Q

From 5’ to 3’ of the mRNA sequence, what is the order of the 3 binding sites of the 50S ribosomal subunit?

A

5’ - E P A - 3’

84
Q

What happens at each of the 3 binding sites?

A

the A site binds an incoming aminoacyl-tRNA

the P site binds a tRNA with a peptide chain and where the elongation occurs (where the aminoacyl group is transferred from the tRNA it came in with to the peptide chain)

the E site is where the uncharged (aminoacyl-free) tRNA is going to be released from the ribosome

85
Q

When does chain elongation begin?

A

after fmet-tRNAfmet (fmet-tRNAfmet) has been bound to the P site

= when the second aminoacyl-tRNA is brought to the A site

86
Q

What are the steps of chain elongation in prokaryotic translation?

A

initiation: fmet-tRNAfmet first occupies the P site of the 50S ribosomal subunit because of the alignment of the codon and anticodon by the Shine-Dalgarno sequence

  1. EF-Tu binds to second aminoacyl-tRNA + GTP and brings tRNA to A site
  2. anticodon of the aminoacyl-tRNA base pairs in an antiparallel fashion with the mRNA codon
    - GTP is hydrolyzed and EF-Tu-GDP complex dissociates
    - EF-Ts regenerates EF-Tu in GTP-bound form
    == new aminoacyl-tRNA bound to A site==
  3. Peptide bond formation reaction catalyzed by peptidyl transferase:
    - alpha amino group of the aminoacyl-tRNA at A site nucleophilic attacks the carbonyl-C of the tRNA at the P site to form the peptide bond (N-C)
    == tRNA at P site is uncharged (no AA), peptide bond formed and now there’s a dipeptidyl-tRNA at A site (growing peptide) ==
  4. Ribosome translocates: ribosome moves along the mRNA (5’-3’) to the next codon
    - uncharged tRNA at P site is moved to E site and released
    - peptidyl-tRNA at A site is moved to P site
    == A site is empty and aligned with the next mRNA codon, ready to accept the new aminoacyl-tRNA ==
87
Q

What enzyme catalyzes the reaction for the peptide bond formation?

A

peptidyl transferase

part of the 50S subunit

88
Q

Describe the peptide bond formation reaction

A

the alpha-amino group (N) of the aminoacyl-tRNA at the A site does a nucleophilic attack on the carbonyl carbon of the peptidyl-tRNA at the P site to form a new peptide bond between the C-N

result: a dipeptidyl-tRNA at the A site and an uncharged (no amino acid) tRNA at the P site

89
Q

Describe translocation

A

before another amino acid can be added to the growing peptide chain:

the uncharged tRNA at the P site moves to the E site where it is released

the peptidyl-tRNA at the A site moves to the P site

the A site is empty and ready to accept a new charged tRNA

the mRNA is moved with respect to the ribosome

GTP hydrolysis is required

90
Q

What are the 3 steps of chain elongation? How often are they repeated? when do they stop?

A
  1. aminoacyl-tRNA binding
  2. peptide bond formation
  3. translocation

they are repeated until there’s a stop signal in the mRNA

91
Q

How is chain elongation terminated? (Ie., what is termination)?

A

chain elongation will continue to occur until the ribosome recognizes a stop signal codon in the mRNA = this is a codon that has no corresponding tRNA

when this occurs, the A site will be empty and the ribosome will be released from the mRNA

92
Q

T or F: chain elongation requires GTP input

A

true

93
Q

What is required for chain termination?

A

a stop signal codon = an mRNA codon that is not recognized by tRNAs/does not have a corresponding tRNA

94
Q

What are the stop codons?

A

UAA, UAG, UGA

95
Q

What recognizes the stop codons?

A

release factors instead of tRNAs

96
Q

T or F: energy input is required for chain termination

A

true, 2 molecules of GTP are needed

97
Q

Describe the steps of termination

A

Stop codon (UAA, UAG, UGA) recognized by release factors

release factors prevent the binding of a new aminoacyl-tRNA and reduce the activity of peptidyl transferase to hydrolyze the peptide bond between the carboxyl of the peptide and the tRNA

GTP is hydrolyzed and the entire ribosomal complex is dissociated

98
Q

What are the major components of amino acid activation in prokaryotic translation?

A

amino acids
tRNAs
aminoacyl-tRNA synthetases
ATP, Mg2+

99
Q

What are the major components of chain initiation in prokaryotic translation?

A

fmet-tRNAfmet
initiation mRNA codon (AUG)
30S ribosomal subunit
50S ribosomal subunit
initiation factors (IF1, IF2, IF3)
GTP, Mg2+

100
Q

What are the major components of chain elongation in prokaryotic translation?

A

70S ribosome
mRNA codons
aminoacyl-tRNAs
elongation factors (EF-Tu, EF-Ts, EF-P, EF-G)
GTP, Mg2+

101
Q

What are the major components of chain termination in prokaryotic translation?

A

70S ribosome
termination mRNA codons (UAA, UGA, UAG)
release factors (RF1, RF2, RF3)
GTP, Mg2+

102
Q

What stages of translation require an energy input?

A

all 4

AA activation requires ATP
initiation, elongation, and termination require GTP

103
Q

T or F: translation is an endergonic reaction

A

true, input of ATP and GTP are required

bioc 299 (16 decks)

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