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Modern Genetic Analysis Chapter 3. Gene Function
Translation

How is amino acid sequence determined? Quite simply, by nucleotide sequence. The specific amino acid sequence of a polypeptide is determined by the nucleotide sequences of the gene that encodes it. The sequence of nucleotides in the DNA of a gene is transcribed into the equivalent sequence in mRNA. Remember that it is the template strand of DNA that is transcribed, but the mRNA synthesized has the same sequence as the nontemplate strand. A phalanx of ribosomes moves along the mRNA, each starting at the 5 end and proceeding along the entire length of the mRNA to the 3 end. As a ribosome moves along, it "reads" the nucleotide sequence of the mRNA three nucleotides at a time. Each group of three, called a triplet codon, stands for a specific amino acid. Since there are four different nucleotides in mRNA, there are 4 × 4 × 4 = 64 different possible codons. These codons and the amino acids they stand for are shown in Figure 3-18. This same genetic code is used by virtually all organisms on the planet. There are some exceptions in which a few of the codons have different meanings.

A Codon Is Translated into an Amino Acid by a tRNA with Complementary Sequence

The basis for the specificity between codon and amino acid lies in the structure of transfer RNA (tRNA) molecules. A molecule of tRNA has a clover-leaf shape consisting of four double-helical stems and three single-stranded loops (Figure 3-19). The middle loop carries a nucleotide triplet called the anticodon, whose job it is to bind with a specific codon in the mRNA by specific RNA-to-RNA base pairing. Since codons in mRNA are read in the 5 3direction, anticodons are oriented in the 3 5 direction, as Figure 3-19 shows. Each tRNA is specific for only one amino acid and carries that amino acid attached at its free 3 end. Amino acids are added to the tRNA by enzymes called aminoacyl-tRNA synthetases. Each amino acid has a specific synthetase that links it to only those tRNAs that recognize the codons for that amino acid. Again we see the principle of nucleic acid complementarity at work, this time in the binding of the codon (RNA) with the anticodon (also RNA).

Figure 3-18 shows that the number of codons for a single amino acid varies, ranging from one (tryptophan, UGG) to as many as six (serine, UCU, UCC, UCA, UCG, AGU or AGC). It is not exactly clear why the code shows this variation, but two facts account for it:

1. Certain amino acids can be brought to the ribosome by several alternative tRNA types having different anticodons, whereas certain other amino acids are brought to the ribosome by only one tRNA.
2. Certain tRNA species can bring their specific amino acids in response to several codons, not just one, through a loose kind of base pairing at the 3 end of the codon and the 5 end of the anticodon. This loose pairing is called wobble.

Wobble is a situation in which the third nucleotide of an anticodon (at the 5 end) can form two alignments (Figure 3-20). This nucleotide can form hydrogen bonds not only with its normal complementary nucleotide in the third position of the codon but also with a different nucleotide in that position. There are "wobble rules" that dictate which nucleotides can and cannot form new hydrogen-bonded associations through wobble (Table 3-2). In the table, the letter I stands for inosine, one of the rare bases found in tRNA, often in the anticodon.

Table 3-3 on the following page lists all the codons for serine and shows how three different tRNAs (tRNASer1, tRNASer2, and tRNASer3) can pair with these codons. In some species there can be an additional tRNA species, which we could represent as tRNASer4, that has an anticodon identical with one of the three anticodons shown in Table 3-3 but differs in its nucleotide sequence elsewhere. These four tRNAs are called isoaccepting tRNAs because they accept the same amino acid, but they are transcribed from different tRNA genes.

Ribosomes Are Protein Factories

The meeting place for amino acidbound tRNAs and mRNA is the ribosome. Ribosomes are large macromolecular assemblies acting like complex subcellular machines. Each ribosome consists of a large and a small subunit. Each subunit in turn is composed of several rRNA types and as many as 50 proteins (Figure 3-21). Ribosomes contain specific sites that enable them to bind to the mRNA, the tRNAs, and other specific protein factors, all required for protein synthesis. Let's look at a general picture of protein synthesis on the ribosome, and then examine each of the steps in the process in more detail.

Figure 3-22 shows a polypeptide being synthesized on the ribosome. The mRNA binds to the small subunit. The tRNAs bind to two sites that overlap the subunits. The A site is the entry site for an aminoacyl-tRNA (a tRNA carrying a single amino acid). The peptidyl-tRNA carrying the growing polypeptide chain binds at the P site. Each new amino acid is added by the transfer of the growing chain to the new aminoacyl-tRNA, forming a new peptide bond. The tRNA that has given up its amino acid is then released from the P site, and the ribosome moves one codon farther along the message, transferring the new peptidyl-tRNA to the P site, and leaving the A site vacant for the next incoming aminoacyl-tRNA. The growing end of the peptide chain (that is, the end on the ribosome) is the carboxyl end, so the free end is the amino end. Hence the amino end of the protein corresponds to the 5 end of the mRNA, and the carboxyl end corresponds to the 3 end.

The action of the ribosome during translation is divided into three distinct steps: initiation, elongation, and termination.

Initiation.

In bacteria, the first amino acid in any newly synthesized polypeptide is N-formylmethionine. It is inserted not by tRNAMet, but by an initiator tRNA called tRNAfMet. This initiator tRNA has the normal methionine anticodon but inserts N-formylmethionine rather than methionine. In E. coli, AUG and GUG, and on rare occasions UUG, serve as initiation codons. When one of these codons occurs in the initiation position, it is recognized by N-formylMet-tRNA and N-formylmethionine appears as the first amino acid in the chain. (The formyl group is lost later.) In eukaryotes AUG is generally the first codon of a polypeptide, but methionine is inserted, not N-formylmethionine.

How are the correct initiation codons selected from the many AUG and GUG codons in an mRNA molecule? In bacteria true initiation codons are preceded by sequences called Shine-Dalgarno sequences that pair well with the 3 end of 16S rRNA in the ribosome, thereby positioning the ribosome properly next to the initiation codon. There is a short but variable separation region between the Shine-Dalgarno sequence and the initiation codon.

Note that in prokaryotes an mRNA has an untranslated "leader sequence" consisting of the RNA between the transcriptional start site and the translational start site (see Figure 3-7). Eukaryotic mRNAs also have a leader sequence but no Shine-Dalgarno sequence; instead, a ribosome carrying an initiator tRNA attaches under guidance of the 5 cap, moves along the RNA, and initiates translation at the first appropriate AUG codon it encounters. In eukaryotes, this codon is determined by certain conserved nucleotides that signal whether a particular AUG is the translation initiation codon, but it is unclear if these sequences are recognized through interactions with ribosomal RNAs.

Elongation.

Elongation is assisted by several proteins, called elongation factors, that guide the binding and movement of tRNAs and the ribosome. The energy to drive the processes is provided by hydrolysis of GTP.

Termination.

You may have noticed in Figure 3-18 that some codons do not specify an amino acid at all. These codons, UAG, UGA, and UAA, are called stop codons or termination codons. They can be regarded as punctuation marks ending the message encoded in the mRNA. Stop codons often are called nonsense codons because they designate no amino acid, but this is misleading because punctuation is very much part of the sense of any message. Interestingly, the three stop codons are not recognized by a tRNA, but instead by protein factors called release factors. When the peptidyl-tRNA is in the P site, the release factors bind to the A site in response to the chainterminating codons. The polypeptide is then released from the P site, and the ribosomes dissociate into two subunits, ending translation.

In one way of analyzing DNA sequences to look for potential genes, computers are programmed to look for open reading frames (ORFs), which are long DNA sequences beginning with an initiation codon (for example 5-ATG-3) and ending with one of the three stop codons.

MESSAGE
Translation is effected by ribosomes moving along mRNA in the 5 3 direction. A set of tRNA molecules brings amino acids to the moving ribosome, their anticodons binding to codons exposed on the ribosome.

© 1999 by W. H. Freeman and Company. All rights reserved.