I. MICROBIAL GENETICS

E. POLYPEPTIDE AND PROTEIN SYNTHESIS

3. Translation

Fundamental statements for this learning object:

1. During translation, specific tRNAs pick up specific amino acids, transfer those amino acids to the ribosomes, and insert them in their proper place according to the mRNA genetic "message."
2. This is done by the anticodon portion of the tRNA molecules complementary base pairing with the codons along the mRNA.
3. Transfer RNA (tRNA) is a three-dimensional, inverted cloverleaf-shaped molecule of RNA.
4. At the top, or 3' end, a specific amino acid can be attached to a specific tRNA by means of specific enzymes called aminoacyl-tRNA synthetases.
5. At the bottom loop of the cloverleaf is a series of three unpaired tRNA bases called the anticodon. An anticodon is a series of three tRNA bases complementary to a mRNA codon.
6. The anticodons of some tRNAs are able to recognize more than one codon because the tRNA's recognition of the third nucleotide of the codon is not always precise, however, the right amino acid is still inserted because there are 61 codons that code for the 22 different amino acids.
7. To initiate translation in prokaryotic cells, a 30S ribosomal subunit binds to a short nucleotide sequence on the mRNA called the ribosome binding site.
8. AUG is known as the start codon. At this point, an initiation complex composed of the 30S subunit, a tRNA having the anticodon UAC and carrying an altered form of the amino acid methionine (N-formylmethionine or f-Met), and proteins called initiation factors is formed. A 50S ribosomal subunit then attaches to the initiation complex and the initiation factors leave. This forms the 70S ribosome.
9. The A or acceptor or aminoacyl site of the ribosome is where an aminoacyl-tRNA first attaches.
10. The P or peptide site of the ribosome is where a tRNA is temporarily holding the growing amino acid chain as the next codon in the mRNA is being read.
11. The E or exit site of the ribosome is where the uncharged tRNA that has released its amino acid exits the ribosome.
12. During peptide bond formation, the amino acid chain or peptide moves from the tRNA at the P-site and forms a peptide bond with the new amino acid attached to the tRNA at the A-site.
13. A stop codon is a series of three mRNA bases coding for no amino acid and thus terminates the protein chain. UAA, UAG, UGA are the three stop codons in the genetic code. (Stop codons do not code for an amino acid because a tRNA cannot recognize them.)
14. A mRNA with multiple ribosomes attached is known as a polyribosome or polysome.

 

LEARNING OBJECTIVES FOR THIS SECTION

---

During translation, specific tRNAs (def) pick up specific amino acids, transfer those amino acids to the ribosomes, and insert them in their proper place according to the mRNA (def) "message." This is done by the anticodon (def) portion of the tRNA molecules complementary base pairing (def) with the codons (def) along the mRNA.

Transfer RNA (tRNA) is a three-dimensional, inverted cloverleaf-shaped molecule of RNA about 70 nucleotides long (see Fig. 1). At the top, or 3' end, a specific amino acid can be attached to a specific tRNA by means of specific enzymes called aminoacyl-tRNA synthetases. The resulting complex of an amino acid and a tRNA is referred to as an aminoacyl-tRNA (def).

At the bottom loop of the cloverleaf is a series of three unpaired tRNA bases called the anticodon. An anticodon (def) is a series of three tRNA bases complementary to a mRNA codon. One loop of the tRNA binds to the aminoacyl-tRNA synthetase charging enzyme while the other loop attaches to the 50S ribosomal subunit. Most of the remaining tRNA bases are involved in intrastrand hydrogen bonds (def) which give the tRNA its specific shape. There are 60 different tRNAs in bacteria.

While there are 64 different mRNA codons, there are no tRNA molecules that possess an anticodon complementary to the three nonsense or stop codons (def). Furthermore, the anticodons of some tRNAs are able to recognize more than one codon because the tRNA's recognition of the third nucleotide of the codon is not always precise. However, the right amino acid is still inserted because there are 61 codons that code for the 22 different amino acids.

If you look again at the genetic code in Fig. 2, you will notice that there are two or more codons coding for every amino acid except methionine. The first two nucleotides of these codons are the same and only the third nucleotide varies. This third nucleotide is where the binding affinity between the tRNA and the mRNA is the weakest and mistakes in translation are most likely to occur. By having several codons coding for the same amino acid, such mistakes in translation often result in the same amino acid being inserted anyway. In addition, when there is a substitution mutation (one nucleotide base is substituted for another by mistake) but two of the three nucleotides in the are still the same, they often code for an amino acid that is very similar to the original one in terms of its ability to be attracted by or repelled by water. This hydrophobicity of amino acids is often critical to a proteins final tertiary structure. By coding for similar amino acids, these mistakes may not affect the final shape and function of the protein significantly.

To initiate translation, a 30S (def) ribosomal subunit (def) binds to a short nucleotide sequence on the mRNA called the ribosome binding site (def). However, translation doesn't usually begin until the 30S ribosomal subunit reaches the first AUG sequence in the mRNA. For this reason, AUG is known as the start codon (def). At this point, an initiation complex (def) composed of the 30S subunit, a tRNA having the anticodon UAC and carrying an altered form of the amino acid methionine (N-formylmethionine or f-Met), and proteins called initiation factors is formed (see Fig. 3).

A 50S ribosomal subunit then attaches to the initiation complex and the initiation factors leave. This forms the 70S ribosome. (see Fig. 4).

The joining of individual amino acids to form a protein or polypeptide is known as the elongation phase of translation. There are three sites on the 70S ribosome. The A or acceptor or aminoacyl site (def) is where an aminoacyl-tRNA (def) first attaches. The P or peptide site (def) is where a tRNA is temporarily holding the growing amino acid chain as the next codon in the mRNA is being read. The E or exit site is where the uncharged tRNA that has released its amino acid exits the ribosome. During peptide bond (def) formation, the amino acid chain or peptide moves from the tRNA at the P-site and forms a peptide bond (def) with the new amino acid attached to the tRNA at the A-site. The peptide bond is formed by a ribozyme (def), an enzyme composed of the 23S rRNA itself, called peptidyl transferase (def). The now uncharged tRNA (def) at the P-site leaves the ribosome through the E-site to eventually pick up a new amino acid and be recycled. Meanwhile, the 70S ribosome moves a distance of one codon down the mRNA through a process called translocation to allow decoding of the next codon in the message (see Fig. 5A - 5F). The growing polypeptide chain actually passes through a tunnel in the 50S ribosomal subunit.

This process continues over and over again in the 5' to 3' direction until the ribosome hits a stop codon. A stop codon (def) is a series of three mRNA bases coding for no amino acid and thus terminates the protein chain. UAA, UAG, UGA are the three stop codons in the genetic code. Stop codons do not code for an amino acid because a tRNA cannot recognize them.

Proteins called release factors (def) free the protein from the tRNA and the two ribosomal subunits come apart to be recycled (see Fig. 5F). During this elongation process, the protein has assumed its three-dimensional functional shape. Proteins called chaperonins assist in the protein folding.

Once the ribosome is clear of the ribosome binding site and the AUG start codon, another 30S ribosomal subunit attaches to the ribosome binding site of the mRNA to initiate another round of translation. In this way, multiple copies of a protein can be produced from a single molecule of mRNA. A mRNA with multiple ribosomes attached is known as a polyribosome or polysome (def).

• QUIZ YOURSELF ON THIS SECTION
• RETURN TO UNIT 7 TABLE OF CONTENTS