Linking RNAs Amino Acids and Ribosomes

As Crick's adapter hypothesis proposed, the translation of mRNA into proteins requires a molecule that links the information contained in mRNA codons with specific amino acids in proteins. That function is performed by tRNA. Two key events must take place to ensure that the protein made is the one specified by mRNA:

► tRNA must read mRNA correctly.

► tRNA must carry the amino acid that is correct for its reading of the mRNA.

Transfer RNAs carry specific amino acids and bind to specific codons

The codon in mRNA and the amino acid in a protein are related by way of an adapter—a specific tRNA with an

12.7 Transfer RNA The tRNA molecule binds to amino acids,asso-ciates with mRNA molecules, and interacts with ribosomes.There is at least one specific tRNA molecule for each of the amino acids.When the tRNA is bonded to an amino acid, it is designated as a charged tRNA.

This computer-generated, space-filling representation shows the three-dimensional structure of a tRNA.

This computer-generated, space-filling representation shows the three-dimensional structure of a tRNA.

Trna Amino Acid Attached

This icon for tRNA will be used in the figures that follow.

This three-dimensional representation emphasizes the internal regions of base pairing.

This icon for tRNA will be used in the figures that follow.

This three-dimensional representation emphasizes the internal regions of base pairing.

Amino Acids Base Pair

Amino acid-attachment site (always CCA)

This flattened "cloverleaf" model emphasizes base pairing between complementary nucleotides.

Amino acid-attachment site (always CCA)

Hydrogen bonds -between paired bases

Hydrogen bonds -between paired bases

11111 y tWT1

The anticodon, composed of the three bases that interact with mRNA, is far from the amino acid attachment site.

The anticodon, composed of the three bases that interact with mRNA, is far from the amino acid attachment site.

tached amino acid. For each of the 20 amino acids, there is at least one specific type ("species") of tRNA molecule.

The tRNA molecule has three functions: It carries (is "charged with") an amino acid, it associates with mRNA molecules, and it interacts with ribosomes. Its molecular structure relates clearly to all of these functions. A tRNA molecule has about 75 to 80 nucleotides. It has a conformation (a three-dimensional shape) that is maintained by complementary base pairing (hydrogen bonding) within its own sequence (Figure 12.7).

The conformation of a tRNA molecule allows it to combine specifically with binding sites on ribosomes. At the 3' end of every tRNA molecule is a site to which its specific amino acid binds covalently. At about the midpoint of tRNA is a group of three bases, called the anticodon, that constitutes the site of complementary base pairing (hydrogen bonding) with mRNA. Each tRNA species has a unique an-ticodon, which is complementary to the mRNA codon for that tRNAs amino acid. At contact, the codon and the anti-codon are antiparallel to each other. As an example of this process, consider the amino acid arginine:

► The DNA coding region for arginine is 3'-GCC-5', which is transcribed, by complementary base pairing, to the mRNA codon 5'-CGG-3'.

► That mRNA codon binds by complementary base pairing to a tRNA with the anticodon 3'-GCC-5', which is charged with arginine.

Recall that 61 different codons encode the 20 amino acids in proteins (see Figure 12.5). Does this mean that the cell must produce 61 different tRNA species, each with a different anti-codon? No. The cell gets by with about two-thirds that number of tRNA species, because the specificity for the base at the 3' end of the codon (and the 5' end of the anticodon) is not always strictly observed. This phenomenon, called wobble, allows the alanine codons GCA, GCC, and GCU, for example, all to be recognized by the same tRNA. Wobble is allowed in some matches, but not in others; of most importance, it does not allow the genetic code to be ambiguous!

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 12.8). 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 + PPj 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 com-ponent—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 ri-bosomes 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 (Figure 12.9). 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 ri-bosomes, 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 cova-lent bonds. If these forces are disrupted by detergents, for example, the proteins and rRNAs separate from one another.

12.8 Charging a tRNA Molecule

Each activating enzyme charges a specific tRNA with the correct amino acid.The enzyme is thus an essential link between the nucleic acid "language" and the protein "language."

The enzyme activates the amino acid, catalyzing a reaction with ATP to form high energy AMP-amino acid and a pyrophosphate ion.

Activating enzyme

The enzyme activates the amino acid, catalyzing a reaction with ATP to form high energy AMP-amino acid and a pyrophosphate ion.

Activating enzyme

Charged tRNA

Activation Amino Acids

Charged tRNA

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 12.9). 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.

mRNA binding site

12.9 Ribosome Structure Each ribosome consists of a large and a small subunit. The subunits remain separate when they are not in use for protein synthesis.

mRNA binding site

12.9 Ribosome Structure Each ribosome consists of a large and a small subunit. The subunits remain separate when they are not in use for protein synthesis.

Ribosomes are irregularly shaped and composed of two subunits.

► 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-tRNA interactions 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.

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Responses

  • yusef
    What matching insures correct amino acids are delivered to the ribosome in the correct order?
    7 years ago

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