Activating enzymes link the right tRNAs and amino acids

The charging of each tRNA with its correct amino acid is achieved by a family of activating enzymes, known more formally as aminoacyl-tRNA synthetases (Figure ). Each activating enzyme is specific for one amino acid and for its corresponding tRNA. The enzyme has a three-part active site that recognizes three smaller molecules: a specific amino acid, ATP, and a specific tRNA. The activating enzyme reacts with tRNA and an amino acid (AA) in two steps:

enzyme + ATP + AA→enzyme—AMP—AA + PPi enzyme—AMP—AA + tRNA→enzyme + AMP + tRNA—AA

The amino acid is attached to the 3′end of the tRNA (to a free OH group on the ribose) with an energy-rich bond, forming charged tRNA. This bond will provide the energy for the synthesis of the peptide bond that will join adjacent amino acids. A clever experiment by Seymour Benzer and his colleagues at the California Institute of Technology demonstrated the importance of the specificity of the attachment of tRNA to its amino acid. In their laboratory, the amino acid cysteine, already properly attached to its tRNA, was chemically modified to become a different amino acid, alanine. Which component— the amino acid or the tRNA—would be recognized when this hybrid charged tRNA was put into a protein-synthesizing system? The answer was: the tRNA. Everywhere in the synthesized protein where cysteine was supposed to be, alanine appeared instead. The cysteine-specific tRNA had delivered its cargo (alanine) to every mRNA “address” where cysteine was called for. This experiment showed that the protein synthesis machinery recognizes the anticodon of the charged tRNA, not the amino acid attached to it. If activating enzymes in nature did what Benzer did in the laboratory and charged tRNAs with the wrong amino acids, those amino acids would be inserted into proteins at inappropriate places, leading to alterations in protein shape and function. The fact that the activating enzymes are highly specific has led to the process of tRNA charging being called the “second genetic code.”

The ribosome is the workbench for translation

Ribosomes are required for the translation of the genetic information in mRNA into a polypeptide chain. Although ribosomes are small in contrast to other cellular organelles, their mass of several million daltons makes them large in comparison with charged tRNAs. Each ribosome consists of two subunits, a large one and a small one. In eukaryotes, the large subunit consists of three different molecules of rRNA and about 45 different protein molecules arranged in a precise pattern. The small subunit consists of one rRNA molecule and 33 different protein molecules. When not active in the translation of mRNA, the ribosomes exist as separated subunits. The ribosomes of prokaryotes are somewhat smaller than those of eukaryotes, and their ribosomal proteins and RNAs are different. Mitochondria and chloroplasts also contain ribosomes, some of which are similar to those of prokaryotes. The different proteins and rRNAs in a ribosomal subunit are held together by ionic and hydrophobic forces, not covalent bonds. If these forces are disrupted by detergents, for example, the proteins and rRNAs separate from one another. When the detergent is removed, the entire complex structure self-assembles. This is like separating the pieces of a jigsaw puzzle and having them fit together again without human hands to guide them! A given ribosome does not specifically produce just one kind of protein. A ribosome can use any mRNA and all species of charged tRNAs, and thus can be used to make many different polypeptide products. The mRNA, as a linear sequence of codons, specifies the polypeptide sequence to be made; the ribosome is simply the molecular workbench where the task is accomplished. Its structure enables it to hold the mRNA and charged tRNAs in the right positions, thus allowing the growing polypeptide to be assembled efficiently.

On the large subunit of the ribosome are four sites to which tRNA binds (see Figure ). A charged tRNA traverses these four sites in order:

- The T (transfer) site is where a charged tRNA first lands on the ribosome, accompanied by a special protein “escort” called the T or transfer factor.

- The A (amino acid) site is where the tRNA anticodon binds to the mRNA codon, thus lining up the correct amino acid to be added to the growing polypeptide chain.

- The P (polypeptide) site is where the tRNA adds its amino acid to the growing polypeptide chain.

- The E (exit) site is where the tRNA, having given up its amino acid, resides before leaving the ribosome and going back to the cytosol to pick up another amino acid and begin the process again. Because codon–anticodon interactions and peptide bond formation occur at the A and P sites, we will describe their function in detail in the next section.

An important role of the ribosome is to make sure that the mRNA–tRNAinteractions are precise: that is, that a charged tRNA with the correct anticodon (e.g., 3′-UAC-5′) binds to the appropriate codon in mRNA (e.g., 5′-AUG-3′). When this occurs, hydrogen bonds form between the base pairs. But these hydrogen bonds are not enough to hold the tRNA in place. The rRNA of the small ribosomal subunit plays a role in validating the three-base-pair match. If hydrogen bonds have not formed between all three base pairs, the tRNA must be the wrong one for that mRNA codon, and that tRNA is ejected from the ribosome.

Translation: RNA-Directed Polypeptide Synthesis

We have been working our way through the steps by which the sequence of bases in the template strand of a DNA molecule specifies the sequence of amino acids in a protein . We are now at the last step: translation, the RNA-directed assembly of a protein. Like transcription, translation occurs in three steps: initiation, elongation, and termination.

Translation begins with an initiation complex

The translation of mRNA begins with the formation of an initiation complex, which consists of a charged tRNA bearingwhat will be the first amino acid of the polypeptide chain anda small ribosomal subunit, both bound to the mRNA. The rRNA of the small ribosomal subunit binds to acomplementary ribosome recognition sequence on themRNA. This sequence is “upstream” (toward the 5′end) ofthe actual start codon that begins translation.Recall that the mRNA start codon in the genetic code isAUG. The anticodon of a methioninechargedtRNA binds to this start codon by complementarybase pairing to form the initiation complex. Thus the firstamino acid in the chain is always methionine. Not all matureproteins have methionine as their N-terminal amino acid. In many cases, the initiator methionine is removedby an enzyme after translation.After the methionine-charged tRNA has bound to the mRNA, the large subunit of the ribosome joins the complex. The methionine-charged tRNA now lies in the P site of the ribosome, and the A site is aligned with the second mRNA codon. These ingredients — mRNA, two ribosomal subunits, and methionine-charged tRNA —are put together properly by a group of proteins called initiation factors.

The polypeptide elongates from the N terminus

A charged tRNA whose anticodon is complementary to the second codon on the mRNA now enters the open A site of the large ribosomal subunit. The large subunit then catalyzes two reactions:

- It breaks the bond between the tRNA in the P site and its amino acid.

- It catalyzes the formation of a peptide bond between that amino acid and the one attached to the tRNA in the A site.

Because the large subunit performs these two actions, it is said to have peptidyl transferase activity. In this way, methionine (the amino acid in the P site) becomes the N terminus of the new protein. The second amino acid is now bound to methionine but remains attached to its tRNA by its carboxyl group (—COOH) in the Asite. How does the large ribosomal subunit catalyze this binding? In 1992, Harry Noller and his colleagues at the University of California at Santa Cruz found that if they removed almost all the proteins in the large subunit, it still catalyzed peptide bond formation. But if the rRNA was destroyed, so was peptidyl transferase activity. Part of the rRNA in the large subunit interacts with the end of the charged tRNA where the amino acid is attached. Thus rRNA appears to be the catalyst. This situation is very unusual because proteins are the usual catalysts in biological systems. The recent purification and crystallization of ribosomes has allowed scientists to examine their structure in detail, and the catalytic role of rRNA in peptidyl transferase activity has been confirmed.

Elongation continues and the polypeptide grows

After the first tRNA releases its methionine, it dissociates from the ribosome, returning to the cytosol to become charged with another methionine. The second tRNA, now bearing a dipeptide, is shifted to the P site as the ribosome moves one codon along the mRNA in the 5′-to-3′direction. The elongation process continues, and the polypeptide chain grows, as the steps are repeated:

- The next charged tRNA enters the open A site.

- Its amino acid forms a peptide bond with the amino acid chain in the P site, so that it picks up the growing polypeptide chain from the tRNA in the P site.

- The tRNA in the P site is released. The ribosome shifts one codon, so that the entire tRNA–polypeptide complex, along with its codon, moves to the newly vacated P site. All these steps are assisted by proteins called elongation factors.

A release factor terminates translation

The elongation cycle ends, and translation is terminated, when a stop codon — UAA, UAG, or UGA — enters the A site. These codons encode no amino acids, nor do they bind tRNAs. Rather, they bind a protein release factor, which hydrolyzes the bond between the polypeptide and thetRNA in the P site. The newly completed protein thereupon separates from the ribosome. Its C terminus is the last amino acid to join the chain. Its N terminus, at least initially, is methionine, as a consequence of the AUG start codon. In its amino acid sequence, it contains information specifying its conformation, as well as its ultimate cellular destination.