26 - Transfer RNA

Question 1

What is Crick’s adaptor hypothesis? How many adaptors are needed for this hypothesis? What is the adaptor in protein synthesis?

Answer 1

An amino acid is carried to the template by an adaptor molecule, and that the adaptor is the part which actually fits onto the RNA.

20 twenty adaptors for this hypothesis, one for each amino acid.

tRNA (transfer RNA) is the adaptor in protein synthesis

Question 2

What is the amino acid attachment site on tRNA?

Answer 2

3’ terminal A residue

Question 3

What is the template recognition site called on tRNA?

Answer 3

The anticodon

Question 4

What proved the adaptor hypothesis?

Answer 4

• A Cysteine tRNA was attached to cysteine
• Raney nickel was used to convert cysteine to alanine
• When this complex was treated with a protein synthesizing system, alanine residues were incorporated into positions normally occupied by Cys residues

This proved that tRNAs are adapted to each amino acids

Question 5

Where are invariant and highly conserved regions on tRNA? (4)

Answer 5

• Surrounding anticodon loop
• In D loop
• In ΤΨC loop
• In acceptor stem

Question 6

Where are variable regions in tRNA? (2)

Answer 6

• D loop

- Variable loop

Question 7

What is type of uracil is found in the ΤΨC loop of tRNA?

Answer 7

Ψ (pseudouridine, aka 5-ribosyluracil)

Question 8

What type of uracil is found in the D loop of tRNA?

Answer 8

dihydrouridine

Question 9

What are two types of unusual base pairings in tRNA?

Answer 9

• Non-standard base pair matches (eg. G=C)

- Base triple interactions (eg. pyridine bound to pyrimidine bound to pyrimidine)

Question 10

What do numbers following bases show in yeast tRNA (eg. A9)?

Answer 10

The position in the sequence of the yeast tRNA

A9: Adenosine at position 9

Question 11

Which type of tRNA structure is variable and which is highly similar?

Answer 11

Variable: secondary structure
Conserved: tertiary structure (L-shaped tertiary structure)

Question 12

What is the critical distance in tertiary tRNA structure?

Answer 12

The distance between the anticodon at one end of the L and the amino acid attached to the 3’ terminus at the other end of the L

Question 13

What is the structure of the amino acid site of tRNA?

Answer 13

An 7 base long acceptor stem followed by:

CCA-OH-3’

The amino acid binds to the 3’ adenine residue

Question 14

Is peptide bond formation thermodynamically favourable or unfavourable? Why?

Answer 14

Thermodynamically unfavourable because it involves the elimination of a water molecule

Question 15

How is the carboxyl (-COOH) group of the precursor amino acid activated? Are there intermediates?

Answer 15

Conversion to an acid anhydride by aminoacyl-tRNA synthetase (ARS)

There is a aminoacyl-adenylate (aminoacyl-AMP) intermediate (mixed anhydride)

Question 16

What tRNA enzyme differs for different amino acids?

Answer 16

aminoacyl-tRNA synthetase (ARS)

Question 17

Write the reaction for the activation of carboxyl groups on amino acids

Answer 17

amino acid + ATP = aminoacyl-AMP + pyrophosphate

Question 18

Amino acids are attached to their appropriate tRNAs by _____?

List the steps of this reaction (2)

Answer 18

aminoacyl-tRNA synthetases

1. Amino acid is recognized by its cognate (specific) aminoacyl-tRNA synthetase and is adenylated
2. Appropriate tRNA is recognized by the ARS and the amino acid residue is transferred to the 2’ or ‘3-OH of the 3’ terminal A residue of the tRNA

Question 19

The high specificity of aminoacyl-tRNA (aa-tRNA) formation by an aminoacyl-tRNA synthetase (ARS) is achieved by what? State the sites where qualities for this activity occur. (3)

Answer 19

Aminoacyl-tRNA synthetases

• High selectivity in recognition of cognate amino acid (at synthetic site)
• High selectivity in recognition of cognate tRNA (tRNA identity elements)
• Proofreading at the stage of the reaction between aminoacyl-adenylate (aa-AMP) and tRNA (hydrolytic site)

Question 20

What enzyme proofreads protein synthesis? How?

Answer 20

Aminoacyl-tRNA synthetase (ARS)

When an incorrect amino acid pairs with ARS, the error is corrected by hydrolysis.

Question 21

What molecule binds between aminoacyl-tRNA synthetase and its cognate amino acid?

Answer 21

AMP

Question 22

What identity elements in tRNA determines the aminoacylation specificity of a tRNA?

Answer 22

A specific collection of single nucleotides and/or base pairs in different tRNAs

Question 23

Anticodon-codon base pairing is _____ and _____. If you have a 3’-CAG-5’ codon, what anticodon binds to this? How would you normally read this codon in a codon bank?

Answer 23

Anticodon-codon base pairing is antiparallel and complementary.

Codon: 3’-CAG-5’
Anticodon: 5’-GUC-3’

This codon would be read 5’-GAC-3’

Question 24

Which nucleotide of the anticodon is called the wobble nucleotide? Why?

Answer 24

The first nucleotide of the anticodon (5’)

This position is flexible in how it paris with the last position of the codon, it is able to interact with more than one kind of base (wobble hypothesis)

Question 25

What is the consequence of a wobble nucleotide?

Answer 25

A single anticodon may be able to recognize more than one codon with codons that differ in the 3rd position (3’)

Question 26

What is the result of deamination of adenine? What can this base pair with (including wobble pairs)

Answer 26

Inosine (I)

Base pairs with adenine, uracil or cytosine

Question 27

Whenever adenosine is in the wobble position (5’ of an anticodon, 3’ of codon) what happens to it?

Answer 27

It is always deaminated to inosine (I) post transcriptionally

Question 28

A tRNA having the anticodon 5’-IGC-3’ is able to decode which codons?

Answer 28

5’-GCU-3’
5’-GCC-3’
5’-GCA-3’

This is due to the wobble effect and adenine in the first anticodon position being deaminated to inosine

Question 29

True or false? RNA frequently contains modified nucleosides? In what circumstance might RNA contain modified nucleosides?

Answer 29

True

Newly synthesized (nascent) RNA contains only standard nucleosides (A, C, U, G), but some of these are modified.

Question 30

What are four characteristics of nucleoside modification in RNA?

Answer 30

• Post-transcriptional
• In a site specific manner
• On either the base, sugar, or both
• By modification enzymes

Question 31

What do tRNA methylases (tRNA methyltransferases) do? What is this an example of?

Answer 31

Transfer methyl groups from a methyl donor (S-adeosylmethionine) into different positions in tRNA

Example of nucleoside modification

Question 32

A given modification enzyme (nucleoside modification in RNA) might be specific for? (3)

Answer 32

• A single site in tRNA
• Several sites in one or more tRNAs
• More than one type of RNA (eg. rRNA and tRNA)

Question 33

What are hypermodifications to nucleosides in RNA?

Answer 33

Chemically complex modifications that are often localized in or adjacent to the anticodon

Question 34

What are four roles of modified nucleosides in tRNA?

Answer 34

• Stabilization of biologically active tRNA conformation
• Stabilization of codon-anticodon interaction
• Expansion or restriction of codon-anticodon pairing (eg. A to I on wobble position)
• Alteration of codon recognition and aminoacylation specificity

Question 35

What is evidence for nucleoside modification’s role in stabilization of biologically active tRNA conformation?

Answer 35

In the absence of specific modifications, a human mitochondrial rRNA(Lys) fails to adopt an active conformation (mitochondrial disease)

Question 36

What does the absence of a hypermodified nucleoside on the 3’ side of the anticodon result in?

Answer 36

The destabilization of codon-anticodon interaction, where tRNA can’t bind stably to mRNA-ribosome complex

Question 37

How does a Ile tRNA accept both AUG (Met) and AUA (Ile)?

Answer 37

The cytidine at the wobble position in a tRNA can be modified to lysidine, changing to anticodon from CAU to LAU. LAU anticodon can accept Ile. (Alteration of codon recognition and aminoacylation specificity by nucleoside modification)