tRNA ACtivation

What is the first major step of translation?

A.a activation

What is the end product of tRNA activation?

Formation of aminoacyl-tRNA

Formation of aminoacyl-tRNA

• the activation of an a.a ocnsists of the formation of an ester bond b/w 3’OH of tRNA (adenosine) and carboxyl group of the AA

  • i.e esterificaiton of AA

• the amino group and side chain remain free (important for elongation process)

• the activation is mediated by the aminoacyl tRNA synthetase (RS)

Aminoacyl-tRNA Synthetase

• for a given AA, there will be one aminoacyl-tRNA synthetase (RS) that activates one or several isoacceptor tRNAs

  • 20 RS

• the synthetase recruits the AA in a binding pocket to bring it near the 3’OH of tRNA

• there are 2 stucturally unrelated classes of aminoacyl-tRNA synthetases

  • Class II enyzymes typically form homodimers

  • they hold tRNAs in diff manners

Aminoacyl-tRNA Synthetase Mech to Activate AA’s

• the aminoacyl tRNA synthetase mediates a 2-step ATP-dependent process

1) Activation of AA: AA + ATP = aminoacyl-AMP (not yet activated) + PPi

• the carboxyl group of the AA, when position correctly in the binding pocket, will attack the ⍺-phosphorus of ATP → to create aminoacyl-AMP

2) activation of tRNA: aminoacyl-AMP + tRNA = aminoacyl-tRNA + AMP

Aminoacyl-tRNA Synthetase Mech to Activate AA’s FIGURE

Differences in the mechs of aminoacyl tRNA synthetase classes

• Class II directly transfers the AA on the 3’OH of the tRNA in step 2B

  • right of the figure

• Class I relies on an additional transesterification (Steps 2a and 3a)

  • left of the figure

  • 2’OH conducts attack → transesterification (AAA is transferred into the 3’OH)

Specificity Challenge of the Genetic Code

• the genetic code defines which AA is added for each codon

• b/c the code is degenerate, multiple codons encode the same AA

• however, maintaining 61 distinct tRNAs and 61 aminoacyl-tRNA synthetases would be energetically costly for the cell

  • enzymes can recognize more than one codon and add the same AA to the tRNA molecule

  • one tRNA can recognize more than one codon

The Second Genetic Code

• the synthetase binds to multiple nucleotides (1-10) on the tRNA but not necessarily via the tRNA anti-codon

  • i.e it recognizes multiple positions on the tRNA, which are scattered across the tRNA structure

• the binding to these tRNA nucleotides is referred to as the 2nd genetic code

  • determines which tRNA will be recruited to a given synthetase, and thereby which AA will be added to that tRNA

“First” vs Second Genetic Code

• First genetic code: codon matched with amino acid (during translation)

• Second genetic code: tRNA matched with amino acid (during tRNA charging)

  • the 2nd code happens before translation begins

• the 2nd genetic code isn’t defined by a simpel table and consists of several residues on the tRNA that mediates their recognition by the aminoacyl-tRNA synthetase

  • it is distinct from codon/anti-codon interaction

Specificity of aminoacyl-tRNA synthetase

• the enzyme places the tRNA in a way so that the 3’OH is located near the AA binding pocket (this is where the AA need to be transferred to get activated tRNA)

Proofreading in tRNA activation: Challenge

• for some AA, creating a specific binding pocket is relatively straightforward, such as for proline, which has a distinct and characteristic structure

  • more challenging for valine vs isoleucine

• AA’s are relatively small molecules

  • if 2 AA are similar, we might activate the incorrect AA’s

  • must ensure high specificity so that the correct AA is added by the RSs

• multiple proofreading mechanisms have evolved that occur at diff steps (pre tRNA transfer or post-tRNA transfer)

Proofreading in tRNA activation: Isoleucyl-tRNA synthetase Solution

• isoleucyl-tRNA synthetase possess two distinct activities

• its active site has a high affinity for Ile and a weak affinity for Val, no affinity for Pro

• the second activity, hydrolysis site exhibits a high affinity Val and low affinity for Ile

  • consequenctly, if Val is incorrectly activated, it will be rapidly hydrolyzed to prevent errors in tRNA charging

Proofreading in tRNA activation: Isoleucyl-tRNA synthetase Solution FIGURE