tRNA Structure, Function, Specificity, Activation

transfer RNA

adaptor in protein synthesis which contains an amino acid attachment site (3’ terminal A residue) and a template recognition site (anticodon)

adaptor hypothesis

model A: adaptor- specificity/recognition on adaptor-amino acid, and specificity/recognition on adaptor-RNA triplet

model B: no adaptor- specificity/recognition of RNA triplet-amino acid

experimental proof of adaptor hypothesis

under reducing conditions (Raney nickel), sulfhydrul (-SH) group of cysteine was chemically converted to alanine, leaving an Ala residue attached to a non-cognate tRNA

when Ala-tRNAcys was subsequently used in a protein-synthesizing system making hemoglobin, Ala residues were incorporated into positions normally occupied by Cys residues

generalized tRNA structure

invariant and highly conserved nucleoside regions (surround anticodon and other areas)

regions that vary in length

anticodon

properties of the tRNA molecule that enable function

has many modified nucleosides

can adopt a precise tridimensional conformation

can establish non-conventional base pairings

unusual base pairings in tRNA

non-standard base pair matches, but functional groups paired in standard ‘Watson-Crick’ pairing

base triple interactions

regardless of their secondary structures which can vary widely, all tRNAs

can be folded into a highly similar tertiary structure

L-shaped tertiary structure of tRNA

critical distance is that 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”

why is peptide bond formation thermodynamically unfavourable?

because it involves elimination of a molecule of water, opposite of hydrolysis

amino acid activation

carboxyl group of precursor amino acid is activated by conversion to an acid anhydride

reaction is catalyzed by an aminoacyl-tRNA synthetase, ARS (a different one activates each of the 20 amino acids found in proteins)

reaction proceeds via the formation of an aminoacyl-adenylate intermediate (aminoacyl-AMP), a mixed anhydride

how are amino acids attached to their appropriate tRNAs?

by aminoacyl-tRNA synthetases, all reactions occur on the ARS

1. amino acid is recognized by its cognate (specific) ARS and is adenylylated

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

high specificity of aminoacyl-tRNA (aa-tRNA) formation by an aminoacyl-tRNA synthetase (ARS) is achieved by

high selectivity in recognition of cognate amino acid (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)

identity elements in tRNAs

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

the codon-anticodon interaction involves

antiparallel, complementary base pairing

• the first 5’ nucleotide of the anticodon pairs with the last 3’ nucleotide of the codon

wobble nucleotide

first nucleotide of the anticodon, this position is flexible in how it pairs with the last position of the codon: it is able to interact with more than one kind of base

wobble hypothesis

enables one tRNA to pair with two or more different codons, results from the properties of RNA that allow noncanonical base pairs to occur

how are newly-synthesized RNA residues modified?

post-transcriptionally, in a site-specific manner, on either the base or sugar (or both), by modification enzymes

a given modification enzyme may be specific for

a single site in a single tRNA molecule

several sites in one or more tRNAs

more than one type of RNA

hypermodifications

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

biosynthesis may require several steps, each step catalyzed by a different modification enzyme

roles of modified nucleosides in tRNA

stabilization of biologically active tRNA conformation

stabilization of codon-anticodon interaction

expansion or restriction of codon-anticodon pairing

alteration of codon recognition and aminoacylation specificity

stabilization of biologically active tRNA conformation example

in the absence of specific modifications, mitochondrial tRNAlys fails to adopt an active conformation→a mitochondrial disease (MERRF)

stabilization of codon-anticodon interaction example

hypermodification nucleosides on the 3’ side of the anticodon

absence of this modification prevents tRNA from binding stably to mRNA-ribosome complex

expansion or restriction of codon-anticodon pairing example

A→I at the first (wobble) position of the anticodon expands pairing from U (with A) to U, C, or A (with I)

modification of wobble U restricts pairing to A only (instead of both A and G)

alteration of codon recognition and aminoacylation specificity example

formation of lysidine (L) from cytidine (C) at wobble position in a tRNAile

tRNAile with CAU anticodon recognizes AUG (Met) and accepts Met

tRNAile with LAU anticodon recognizes AUA (Ile) and accepts Ile