26 - Transfer RNA Flashcards
585-590
What is Crick’s adaptor hypothesis? How many adaptors are needed for this hypothesis? What is the adaptor in protein synthesis?
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
What is the amino acid attachment site on tRNA?
3’ terminal A residue
What is the template recognition site called on tRNA?
The anticodon
What proved the adaptor hypothesis?
- 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
Where are invariant and highly conserved regions on tRNA? (4)
- Surrounding anticodon loop
- In D loop
- In ΤΨC loop
- In acceptor stem
Where are variable regions in tRNA? (2)
- D loop
- Variable loop
What is type of uracil is found in the ΤΨC loop of tRNA?
Ψ (pseudouridine, aka 5-ribosyluracil)
What type of uracil is found in the D loop of tRNA?
dihydrouridine
What are two types of unusual base pairings in tRNA?
- Non-standard base pair matches (eg. G=C)
- Base triple interactions (eg. pyridine bound to pyrimidine bound to pyrimidine)
What do numbers following bases show in yeast tRNA (eg. A9)?
The position in the sequence of the yeast tRNA
A9: Adenosine at position 9
Which type of tRNA structure is variable and which is highly similar?
Variable: secondary structure
Conserved: tertiary structure (L-shaped tertiary structure)
What is the critical distance in tertiary tRNA structure?
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
What is the structure of the amino acid site of tRNA?
An 7 base long acceptor stem followed by:
CCA-OH-3’
The amino acid binds to the 3’ adenine residue
Is peptide bond formation thermodynamically favourable or unfavourable? Why?
Thermodynamically unfavourable because it involves the elimination of a water molecule
How is the carboxyl (-COOH) group of the precursor amino acid activated? Are there intermediates?
Conversion to an acid anhydride by aminoacyl-tRNA synthetase (ARS)
There is a aminoacyl-adenylate (aminoacyl-AMP) intermediate (mixed anhydride)
What tRNA enzyme differs for different amino acids?
aminoacyl-tRNA synthetase (ARS)
Write the reaction for the activation of carboxyl groups on amino acids
amino acid + ATP = aminoacyl-AMP + pyrophosphate
Amino acids are attached to their appropriate tRNAs by _____?
List the steps of this reaction (2)
aminoacyl-tRNA synthetases
- Amino acid is recognized by its cognate (specific) aminoacyl-tRNA synthetase and is adenylated
- 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
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)
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)
What enzyme proofreads protein synthesis? How?
Aminoacyl-tRNA synthetase (ARS)
When an incorrect amino acid pairs with ARS, the error is corrected by hydrolysis.
What molecule binds between aminoacyl-tRNA synthetase and its cognate amino acid?
AMP
What identity elements in tRNA determines the aminoacylation specificity of a tRNA?
A specific collection of single nucleotides and/or base pairs in different tRNAs
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?
Anticodon-codon base pairing is antiparallel and complementary.
Codon: 3’-CAG-5’
Anticodon: 5’-GUC-3’
This codon would be read 5’-GAC-3’
Which nucleotide of the anticodon is called the wobble nucleotide? Why?
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)
What is the consequence of a wobble nucleotide?
A single anticodon may be able to recognize more than one codon with codons that differ in the 3rd position (3’)
What is the result of deamination of adenine? What can this base pair with (including wobble pairs)
Inosine (I)
Base pairs with adenine, uracil or cytosine
Whenever adenosine is in the wobble position (5’ of an anticodon, 3’ of codon) what happens to it?
It is always deaminated to inosine (I) post transcriptionally
A tRNA having the anticodon 5’-IGC-3’ is able to decode which codons?
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
True or false? RNA frequently contains modified nucleosides? In what circumstance might RNA contain modified nucleosides?
True
Newly synthesized (nascent) RNA contains only standard nucleosides (A, C, U, G), but some of these are modified.
What are four characteristics of nucleoside modification in RNA?
- Post-transcriptional
- In a site specific manner
- On either the base, sugar, or both
- By modification enzymes
What do tRNA methylases (tRNA methyltransferases) do? What is this an example of?
Transfer methyl groups from a methyl donor (S-adeosylmethionine) into different positions in tRNA
Example of nucleoside modification
A given modification enzyme (nucleoside modification in RNA) might be specific for? (3)
- A single site in tRNA
- Several sites in one or more tRNAs
- More than one type of RNA (eg. rRNA and tRNA)
What are hypermodifications to nucleosides in RNA?
Chemically complex modifications that are often localized in or adjacent to the anticodon
What are four roles of modified nucleosides in tRNA?
- 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
What is evidence for nucleoside modification’s role in stabilization of biologically active tRNA conformation?
In the absence of specific modifications, a human mitochondrial rRNA(Lys) fails to adopt an active conformation (mitochondrial disease)
What does the absence of a hypermodified nucleoside on the 3’ side of the anticodon result in?
The destabilization of codon-anticodon interaction, where tRNA can’t bind stably to mRNA-ribosome complex
How does a Ile tRNA accept both AUG (Met) and AUA (Ile)?
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)
