Chapter 9 – Translation

“The synthesis of every protein molecule in a cell is directed by that cell’s DNA”

There are two aspects to how this is accomplished:

• There is the information or coding problem: How does the sequence of nucleotides specify the sequence of amino acids?
• There is the chemistry problem: What is the actual process by which the different amino acids are linked together in the correct sequence to form a functional protein?

Introduce the Players and their roles:

• Ribosomes – These complex organelles are composed of several pieces of rRNA and many proteins. These organelles are the actual “factories” where protein synthesis takes place.

• mRNA – this is the coded information for the protein which is about to be made. This information is copied from the DNA template. That template is called the gene for that protein.

• tRNA – This is the adapter molecule that links the language of mRNA to the language of proteins. These RNA molecules have an amino acid attachment site as well as an “anti-codon” that can attach specifically to the three-base-long “codon” found on the mRNA.

See fig. 9-1 for an overview of how translation occurs. Note that there is a direction: the mRNA is read 5’ à 3’.

Also note that the new protein (nascent protein) is made amino-terminus first.

Please see figures 9-2 and 9-3 for a few more specific details:

• the relationship of the template strand of DNA to the message
• the definition of “codon”

The Genetic Code – Table 9-1

• AUG is the start codon (sometimes GUG)
• Methionine and tryptophan have only one codon each. All the other amino acids have several codons – note that usually it is the third base that is different. This is often referred to as the “degeneracy” of the genetic code
• Stop codons don’t code for amino acids. These are stop codons because there is no tRNA that matches them.
• Note that this table depicts the mRNA codons written 5’ à 3’. We could just as appropriately devise a table of anti-codons written 3’ à 5’

What is tRNA? – See fig 9-4 and 9-5.

The tRNA molecule has several important characteristics:

• The tRNA molecule has an “anticodon”
• The tRNA molecule has an “amino acid attachment site at its 3’-end
• An “amino-acyl-tRNA-synthetase” (specific for each tRNA/amino acid combination) matches the amino acid to the anti-codon and attaches the amino acid. (The enzyme charges or acylates the tRNA.)

See a movie describing how tRNA gets acylated

• When the tRNA binds to the mRNA, the binding to the 3^rd position of the codon is “wobbly.” (See table 9-2. Note that the unusual purine base “inosine” is often present in the third position of the anticodon – this base can pair with U, C, and A.)
• This explains why the code is degenerate.

Let’s review the general structure of mRNA

Let’s review polycistronic RNA again:

• See figure 9-6.
• Note that each of the protein coding regions (cistrons) starts with an AUG start codon.
• Note that each cistron also ends with a stop codon
• Note that there are ‘spacer” sequences between each of the protein coding regions
• Note that there is a “leader” sequence at the beginning of the message, before the first start codon.

What are “overlapping” genes?

The book refers to the phage fX174 – a single-stranded DNA virus.

· Analysis revealed that the genome of this virus was too small to account for all the proteins that it could make.

· The problem was solved by finding that some of fX174’s genes are overlapping:

o The same nucleotide sequence could be read in multiple reading frames:

o The sequence below could be read differently depending on which AUG is used as a start codon:

AUGNNNNAUGNNNNNNNAUGNN… … …

AUG NNN NAU GNN NNN NNA UGN N.. or

AUG NNN NNN NAU GNN … or

AUG NN.

· Also see fig 9-7.

Polypeptide Synthesis

· Prokaryotic ribosome structure – 70s ribosomes

· 30s subunit – 16s rRNA plus 21 polypeptides

· 50s subunit – 23s rRNA, 5s rRNA and 32 polypeptides

· Eukaryotic ribosome structure – 80s ribosomes

· 40s subunit – 18s rRNA plus 30 proteins

· 60s subunit – 5s rRNA, 5.8s rRNA, 28s rRNA and 50 proteins

· Initiation

· The 16s rRNA of the 30s subunit binds to the “Shine-Delgarno” sequence.

§ The Shine-Delagarno sequence (AGGAGGU) is also known as the “translation initiation region.

§ The eukaryotic counterpart is the 5’-cap of the eucaryotic message.

§ (See fig 9-11) The “pre-initiation complex” forms: the 30s subunit + mRNA + f-met-tRNA + initiation factors (proteins) + GTP.

§ Then the 50s subunit binds and “elongation” begins.

· Note the A-site and the P-site in the figure

· Elongation (fig. 9-13)

· With f-met-tRNA in the P-site and the next acylated-tRNA in the A-site ---- a peptidyl transferase forms a peptide bond between the two amino acids.

· F-met is cleaved from its tRNA and the tRNA leaves, leaving the P-site open.

· The second tRNA “translocates” into the P-site from the A-site.

· A new acylated tRNA moves into the empty A-site to base-pair with the codon in that site.

· This process repeats over and over until a nonsense codon is presented in the A-site.

· Termination

· Release factors cleave everything is the A-site is unoccupied for too long.

· In polycistronic mRNA, the next AUG is not too far away and the ribosome reinitiates to synthesize the next protein.

What are polysomes?

• Please study figures 9-15 and 9-16

Read about Antibiotics and ribosomes on page 188.