Genetics Notes
Chapter 15 - The Genetic Code and Translation

These notes are provided to help direct your study from the textbook. They are not designed to explain all aspects of the material in great detail; that is what class time and the textbook is for. If you were to study only these notes, you would not learn enough genetics to do well in the course.

Translation is the process of creating a polymer of amino acids (protein) using mRNA to direct the order of addition.

Transfer RNA The function of tRNA is to ensure that each amino acid incorporated into a protein corresponds to a particular codon on the messenger RNA. tRNA does this by having an anti-codon in one of its loops, which binds to the codon on the mRNA, and an amino acid at the 3' end of the tRNA (figure 15.17). The correct amino acid is attached to the correct tRNA by an enzyme called aminoacyl-tRNA synthetase (Figure 11.6).
1) amino acid (aa) + ATP ---> aa-P-adenosine + PiPi    (Pi is a phosphate group)
2) aa-P-adenosine + tRNA ---> aa-tRNA + adenosine-Pi

The amino acid adds at the 3'-OH group of the adenine of the 3' end of the tRNA. This bond is a high energy bond.

Each synthetase is specific for a particular amino acid and its tRNA. However, occasional mistakes are made. Isoleucyl-tRNAile synthetase will attach valine about 1 in 225 times. However, there is a proofreading step during which the incorrect amino acid is removed. Only 1/270 to 1/800 mistakes go uncorrected. This results in 1 incorrectly charged tRNA per 60,000 to 80,000 formed.

There are about 50 tRNA's known in bacteria. The minimum is 20 (1 for each amino acid). The maximum is 61 (1 for each possible codon). The difference is due to the fact that some anti-codons can bind to more than one codon. The is called the "wobble" phenomenon and occurs in the third position of the codon.

Recognition of aminoacyl-tRNA
During protein synthesis, is the amino acid the structure that is recognized or is it the tRNA to ensure the correct sequence of amino acids?

Chapeville did a simple experiment to test this. He isolated cysteine-tRNAcys and removed the SH group, which yielded alanine-tRNAcys. When this tRNA was used during protein synthesis, an alanine was put in the protein chain where cysteine should have been. This showed that the tRNA is the part that is recognized by its binding to the codon on the mRNA and that the amino acid plays no role in recognition.

Initiation
It is very important that translation start at the right place, otherwise the amino acid sequence could be very garbled and a non-functional protein would result (figure 15.20).

The synthesis of every protein in E. coli begins with the amino acid N-formyl methionine, which is then removed before the protein becomes functional. Methionine has the codon 5'-AUG-3'. This codon has two tRNA's that are complementary to it. One of the tRNAs (tRNAfmet) serves as part of the initiation complex. The other (tRNAmet) will have methionine attached and will be used for protein synthesis when methionine is added at later positions in the growing peptide chain.

Initiation complex
In prokaryotes, the subunits (50S and 30S) of the ribosome are disassociated when not involved in translation. The initiation complex (figure 15.19) contains:
1) 30S subunit
2) mRNA
3) N-formyl methionine tRNAfmet
4) 3 initiation factors - IF1, IF2, and IF3
5) GTP
6) 50S subunit

Formation 1
The prokaryotic mRNA is recognized by the ribosome and IF3 due to binding of a complementary region at the 3' end of the 16S rRNA upstream from the initiation codon on the mRNA. This region of the mRNA is called the Shine-Dalgarno sequence (figure 15.19).

Formation 2 is formed by IF-2, GTP and fmet-tRNAfmet. These along with IF-1 bind to Formation 1 (figure 15.19).

Finally, the 50S subunit binds such that a fully functional ribosome is assembled and it is assembled at the correct place on the mRNA (Figure 11.11).

The IF factors are released. The hydrolysis of the GTP is to provide energy for the conformational changes in the proteins to facilitate release of the initiation factors.

The P site, A site, and E site are fully formed at this time. The P site is the site where the growing peptide chain will sit. The A site is the site where each subsequent aminoacyl-tRNA will bind. The E site is where the used tRNA sits before it moves to the cytoplasm (figure 15.19).

Elongation
To begin elongation requires the correct tRNA, GTP and two proteins (elongation factors EF-Ts and EF-Tu) (figure 15.22).
1) EF-Tu.EF-Ts complex binds GTP
2) EF-Ts is released and EF-Tu.GTP complex forms
3) EF-Tu.GTP complex reacts with the aminoacyl-tRNA
4) the EF-Tu.GTP.aa-tRNA complex binds to the A site, GTP is hydrolyzed to GDP and Pi and the EF-Tu can then reform the EF-Tu.EF-Ts complex

Peptidyl transferase transfers the peptide chain from the tRNA at the P site to the amino group of the amino acid of the tRNA at the A site (figure 15.22).

The next step is translocation of the tRNA with the peptide chain from the A site to the P site. The process of translocation involves moving the entire ribosome down 3 nucleotides. In the process, the depleted tRNA at the P site is moved to the E site before being ejected to the cytoplasm and the tRNA-peptide is moved to the P site. This leaves the A site open for the next tRNA. This process requires GTP and an elongation factor (EF-G) sometimes called translocase. The hydrolysis of the GTP to GDP and Pi allows the release of EF-G. The process repeats itself until termination occurs (figure 15.22).

The energy cost for protein synthesis is very high. Two GTP are used in the direct formation of the peptide bonds. One ATP is converted to AMP and PiPi when the tRNA is charged. This is equivalent to two more high energy bonds. An average protein of 300 amino acids requires the equivalent of 1200 ATP's for peptide bond formation. In E. coli, 90% of its energy is spent on protein synthesis. The average speed of production is 20 peptide bonds per second.

Termination
Termination occurs when one of three nonsense codons appears in the A site of the ribosome. They are UAG, UAA, and UGA. When the termination codon appears in the A site, it is recognized by a release factor (figure 15.23).
RF-1 - UAA, UAG RF-2 - UAA, UGA

1) a third release factor RF-3 is required for the breaking of the linkage of the terminal amino acid to its tRNA to release the peptide 2) GTP is hydrolyzed to GDP + Pi 3) mRNA is released 4) releasing factors disassociate 5) disassociation of the ribosome is aided by one of the original (IF-3) initiation factors
In prokaryotes, the majority of mRNAs contains the information for several genes. These RNAs are termed polycistronic. Each gene, however, is translated independently, each has its own initiation sequences, etc (figures 15.25 and 15.26). In eukaryotes, every mRNA contains only one gene, so they are monocistronic. Because each mRNA needs the 5' cap for recognition by the ribosome, you can only have one initiation site per RNA in eukaryotes.

The genetic code
     
    transcription       translation
DNA --------------> RNA -----------> protein
What is the genetic code? How is information that is coded in the DNA used to direct protein synthesis?

In the experimental procedures used to determine the genetic code, RNA was used, so the genetic code is given in terms of RNA. As RNA is exactly complementary to DNA, you can use either DNA codons or RNA codons. A codon is the coding unit, or the specific base sequence that codes for an amino acid.

Length of a codon
How many bases are needed to code for an amino acid? If a codon was one base long, then four amino acids could be coded for. If a codon was 2 bases long = 42 = 16 amino acids could be coded for. If a codon was 3 bases long = 43 = 64 amino acids could be coded for. Therefore, at least 3 bases were needed to code for the 20 amino acids needed for protein synthesis.

To determine the length of a codon, Crick and his coworkers exposed a T4 phage to a chemical agent that deletes or adds single base pairs. The complete amino acid sequence of the protein was worked out and in the pseudowild type, the protein was found to have a group of five amino acid not found in the wild type.
Results can be interpreted accordingly
   C A T   C A T   C A T   C A T   C A T      

             delete one base
   C A T   A T C   A T C   A T C   A T C  (frame is shifted one base)

              add new base
   C A T   A T C   A T G   C A T   C A T  (reading frame is restored,
        but there are 2 non-normal amino acids)
They found that if 3 nucleotides were added or 3 nucleotides were deleted, the reading frame was maintained. They then concluded that a codon was a triplet, or consists of 3 bases. These studies also indictaed that there was no internal punctuation or overlap in the genetic code.

In 1961, Nirenberg and Ochoa produced an artificial RNA composed entirely of uracil, poly(U). Nirenberg and Matthaei took artificail mRNA and were able to synthesize a protein in a cell-free preparation of ribosome. This was found to code for a protein that was all phenylalanine (figure 15.9).

The other codons were worked out using a statistical analysis of the predicted ratios of codons from synthesized RNA from specific ratios of nucleotides. For example, poly(UG), in which U is twice as common as G, would code for leucine (UUG), valine (GUU), or cysteine (UGU) (figure 15.10). Even though they could not make precise statements about which codon went with which amino acid, they could rule out many possibilities.

Eventually, it became possible to make pieces of RNA with the exact sequence of bases. Nirenberg and Leder used an assay system that consisted of
1) a synthetic trinucleotide
2) ribosomal preparation from E. coli
3) aminoacyl-tRNA in which the amino acid is radioactively labeled
4) high molar Mg
This was called a trinucleotide binding assay (figure 15.11). They performed a series of experiments that determined absolutely which codon went with each amino acid. By 1967, all 64 codons had been worked out using these synthetic RNAs.
61 specified amino acids
3 were termination codons
1 was an initiation codon, and also coded for methionine.

Wobble hypothesis
There are only 20 amino acids that are coded for by approximately 50 different tRNA and there are 61 codons that specify an amino acid. As it turns out, some tRNA's can bind at more than one codon. Crick called this ability "wobble." If inosine is in the first position in the anticodon of the tRNA, it can bind to uracil, adenosine, or cytosine in the third position of the codon. Thus this one tRNA recognizes three different codons (figures 15.12 and 15.13). Remember that the codon and anti-codon are anti-parellel. The first position of the codon is at the 5' end of the codon and binds to the third position (3'end) of the anti-codon.

In summary,
1) the code is a triplet. Each codon consists of a unique combination of 3 nucleotides.
2) the code has punctuations only at the ends (start and stop). Start codon is AUG. Stop codons are UAA, UAG, and UGA.
3) most amino acids are specified by more than one codon, so the code is redundant. For example, UCU, UCC, UCA, UCG, AGU, and AGC all code for serine. This is true for 18 of the 20 amino acids. Only methionine and tryptophan are specified by a single codon.
4) the code is consistent. Each one of the codons specifies a unique amino acid.
5) the code is universal. Viruses, bacteria, plants, and animals all use the same code. There are some minor variations in mitochondria and a few fungi.

Last update on 25 November 2004
Provide comments to Dwight Moore at mooredwi@emporia.edu
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