Lecture 6 Bio 99

Identity nucleotides

tRNA nucleotides that are an ID tag for correct aats. scattered throughout tRNA.

Why can one aminoacyl-tRNA synthetase recognize multiple tRNAs?

tRNAs for the same amino acid share the same "ID tags," so one synthetase can load them all with that amino acid.

How does the Ile-tRNA synthetase prevent Valine from getting

attached to the tRNA Ile?

It cannot, so it needs to proofread when aa are structurally similar.

AA-tRNA Proofreading

1. Acylation site

If Amino Acid is too big, it is immediately rejected and hydrolyzed from end of tRNA before releasing from synthetase.

2. Proofreading Site

The amino acid comes here if it is small and fits in acylation site. Proofreading site is made to be too small for the correct amino acid. So if the attached amino acid fits into the site, it is wrong, and is hydrolyzed and released.

Bacteria Initiator Met

tRNA^fmet

Bacteria Internal Met

tRNA^met

What makes fmet?

Transformylase converts methionine

f-Met

Bacterial initiator Methionine

• Formyl group is attached to the N-terminus (like 5’), so it cannot attach any amino acid to that site, meaning it can only be the beginning of an amino acid sequence, and cannot be added internally.

• Only tRNA recognized by ribosome initiation complex.

Eukaryote Initiator

tRNA_I^Met , Not modified like fMet, the only one recognized by ribosome intiator complex.

Eukaryote Internal

tRNA^Met

Aminopeptidases often remove the N-terminal Met, so many mature proteins don’t have Met as the first AA

tRNA structure

anticodon arm, amino acid arm, other arms

Anticodon Arm

Has the anticodon that pairs with the mRNA codon.

Amino Acid Arm

Attaches to the amino acid, opposite from anticodon arm. The 3’CCA of the tRNA is what is attached to the amino acid.

tRNA^tyr

Recognizes tyrosine

Tyr-tRNA^tyr

Charged with tyrosine.

Aminoacyl-tRNA

Charged tRNA

Why does tRNA need to be processed?

tRNA is not ready to use immediately after it is transcribed. It must be modified and trimmed before becoming a functional tRNA.

What needs to happen to process tRNA?

• Base modification

• Cleavage of ends.

• CCA addition

• Intron removal in eukaryotes

tRNA Activation

1. Adenylylation

Amino Acid + ATP—> aminoacyl-AMP + PPi

AMP attaches to Carboxyl group of amino acid, and PPi provides energy to drive and reverse this rxn.

2. tRNA Charging

Aminoacyl-AMP + tRNA —> Aminoacyl-tRNA + AMP

Aminoacyl is transferred off of AMP onto tRNA CAA arm.

2 classes of AATS charge differently.

AATS considered “second genetic code”.

Class 1 AATS

Attach amino acid onto 2’OH of CCA. (On the A).

• Will spontaneously switch from 2’OH to 3’OH, so in both classes, the amino acid will end up on the 3’OH.

Class 2 AATS

Attach amino acid onto 3’OH of CCA