October 16, 2012

tRNAs are charged with amino acids by synthetases

  • Cognate tRNAs (Isoaccepting tRNA) are those recognized by a particular aminoacyl-tRNA synthetase. They all are charged with the same amino acid.
  • Aminoacyl-tRNA synthetases are enzymes that charge tRNA with an amino acid to generate aminoacyl-tRNA in a two-stage reaction that uses energy from ATP.
  • There are 20 aminoacyl-tRNA synthetases in each cell. Each charges all the tRNAs that represent a particular amino acid.
  • Recognition of a tRNA is based on a small number of points of contact in the tRNA sequence. 

It is necessary for tRNAs to have certain characteristics in common, yet be distinguished by others. The crucial feature that confers this capacity is the ability of tRNA to fold into a specific tertiary structure. Changes in the details of this structure, such as the angle of the two arms of the "L" or the protrusion of individual bases, may distinguish the individual tRNAs.
All tRNAs can fit in the P and A sites of the ribosome, where at one end they are associated with mRNA via codon-anticodon pairing, while at the other end the polypeptide is being transferred. Similarly, all tRNAs (except the initiator) share the ability to be recognized by the translation factors (EF-Tu or eEF1) for binding to the ribosome. The initiator tRNA is recognized instead by IF-2 or eIF2. So the tRNA set must possess common features for interaction with elongation factors, but the initiator tRNA can be distinguished.

Amino acids enter the protein synthesis pathway through the aminoacyl-tRNA synthetases, which provide the interface for connection with nucleic acid. All synthetases function by the two-step mechanism depicted in Figure 7.13:
  • First, the amino acid reacts with ATP to form aminoacyl~adenylate, releasing pyrophosphate. Energy for the reaction is provided by cleaving the high energy bond of the ATP.
  • Then the activated amino acid is transferred to the tRNA, releasing AMP.
The synthetases sort the tRNAs and amino acids into corresponding sets. Each synthetase recognizes a single amino acid and all the tRNAs that should be charged with it. Usually, each amino acid is represented by more than one tRNA. Several tRNAs may be needed to respond to synonym codons, and sometimes there are multiple species of tRNA reacting with the same codon. Multiple tRNAs representing the same amino acid are called isoaccepting tRNAs; because they are all recognized by the same synthetase, they are also described as its cognate tRNAs.
Many attempts to deduce similarities in sequence between cognate tRNAs, or to induce chemical alterations that affect their charging, have shown that the basis for recognition is different for different tRNAs, and does not necessarily lie in some feature of primary or secondary structure alone. We know from the crystal structure that the acceptor stem and the anticodon stem make tight contacts with the synthetase, and mutations that alter recognition of a tRNA are found in these two regions. (The anticodon itself is not necessarily recognized as such; for example, the "suppressor" mutations discussed later in this chapter change a base in the anticodon, and therefore the codons to which a tRNA responds, without altering its charging with amino acids.)
A group of isoaccepting tRNAs must be charged only by the single aminoacyl-tRNA synthetase specific for their amino acid. So isoaccepting tRNAs must share some common feature(s) enabling the enzyme to distinguish them from the other tRNAs. The entire complement of tRNAs is divided into 20 isoaccepting groups; each group is able to identify itself to its particular synthetase.
tRNAs are identified by their synthetases by contacts that recognize a small number of bases, typically from 1-5. Three types of feature commonly are used:
  • Usually (but not always), at least one base of the anticodon is recognized. Sometimes all the positions of the anticodon are important.
  • Often one of the last three base pairs in the acceptor stem is recognized. An extreme case is represented by alanine tRNA, which is identified by a single unique base pair in the acceptor stem.
  • The so-called discriminator base, which lies between the acceptor stem and the CCA terminus, is always invariant among isoacceptor tRNAs.
No one of these features constitutes a unique means of distinguishing 20 sets of tRNAs, or provides sufficient specificity, so it appears that recognition of tRNAs is idiosyncratic, each following its own rules.
Several synthetases can specifically charge a "minihelix" consisting only of the acceptor and Tψ C arms (equivalent to one arm of the L-shaped molecule) with the correct amino acid. For certain tRNAs, specificity depends exclusively upon the acceptor stem. However, it is clear that there are significant variations between tRNAs, and in some cases the anticodon region is important. Mutations in the anticodon can affect recognition by the class II Phe-tRNA synthetase. Multiple features may be involved; minihelices from the tRNAVal and tRNAMet (where we know that the anticodon is important in vivo) can react specifically with their synthetases.
So recognition depends on an interaction between a few points of contact in the tRNA, concentrated at the extremities, and a few amino acids constituting the active site in the protein. The relative importance of the roles played by the acceptor stem and anticodon is different for each tRNA·synthetase interaction (for review see Schimmel, 1989).