Translation

hnRNP = heterogeneous nuclear ribonucleoproteins

PABP = polyadenosine binding protein

I. Nuclear export – mRNA has to meet certain criteria to be exported. This applies to the majority of mRNA in eukaryotes. (see below)

1. Characteristics of mature mRNA ready to be exported.

a. Spliced – contains no introns

i. hnRNPs that bind introns keep the un-spliced mRNA in the nucleus, when those hnRNPs are absent from the mRNA they are able to be exported.

b. Contains export hnRNPs

i. hnRNPs that bind the exons aid in the export of the spliced mRNA.

c. Contains PABPs

d. Has the 5’ cap complex.

2. Nuclear pore – nuclear export of mRNA occurs through the nuclear pore complex. Thought to occur through protein-nuclear pore complex interactions.

II. The genetic code

1. Codon – coding RNA is read in codons which are three consecutive nucleotides. Each codon translates into one amino acid. (see below)

2. Reading frames – since a codon consists of three nucleotides, the mRNA can be translated into one of three reading frames. (see below)

III. Transfer RNA (tRNA) – small RNA adapter molecules.

1. Structure

a. Composed of three loops and a 3’ tail. (see below)

i. The anticodon loop contins the anticodon which is the reverse compliment of the codon.

ii. The tail is covalently bound to the amino acid.

2. Generation of tRNA/amino acid complex

a. The carbonyl group on amino acids are covalently linked to the 3’ hydroxyl group of the tRNA

b. This modification is catalyzed by aminoacyl-tRNA synthetases (see below)

i. This requires ATP energy this energy is stored for the creation of the peptide bond.

ii. In eukaryotes there are 20 synthetases one for each amino acid

iii. In prokaryotes there are fewer than 20 synthetases but uses enzymes to chemically modify the amino acids that are matched up to the wrong codon into the correct amino acid.

iv. Synthetases recognize both the amino acid and the tRNA anticodon.

III. Ribosome – guides the synthesis of proteins by joining the tRNA with the mRNA and creating the polypeptide.

1. Structure

a. Composed of proteins and ribosomal RNA (rRNA)

b. Has two subunits; a large and a small subunit that are different between pro- and eukaryotes (see below)

c. the large subunit has three sites important for translation; A, P, E sites (see elongation)

IV. Translation initiation

1. Prokaryotic (see below)

1. The initiation tRNA has an anticodon for the AUG codon and has a formylmethionine

2. Prokaryotic mRNA do not have a 5’ cap to imitate translation so the use a Shine-Dalgarno sequence.

3. Shine-Dalgarno sequence

i. 5’AGGAGGUXXXXXXXXXAUG 3’

ii. binds the 16S rRNA

4. Because the ribosome can assemble right on the start sequence, prokaryotic mRNAs are often polycistronic meaning one mRNA can encode for several different proteins.

2. Eukaryotic

a. The initiation tRNA has an anticodon for the AUG codon and has a methionine

b. Cap dependant (90% of mRNA) - small ribosome subunit assembly occurs at the 5’ cap complex. (see below)

c. Cap independent (10% of mRNA) - IRES (internal ribosome entry sequence)

i. IRESs are several hundred base pairs and allow for ribosomal assembly.

ii. Common in viral mRNA. Some viruses cleave eIF4G thereby stopping cap dependant translation. Because they have IRES in their mRNA they are immune to this shut down.

V. Elongation

1. Three steps that cycle until termination (see below):

1. tRNA carrying the next amino acid in the peptide sequence binds the A site of the ribosome via codon-anticodon interactions.

2. The high energy bond between carbonyl of the amino acid and the tRNA ribose drives the formation of the peptide bond between the amino acid in the P site and the amino acid in the A site. The acive site on the ribosome that does this is said to have peptidyl transferase activity.

3. There is a conformational change in the ribosome that allows the mRNA to be shifted over exactly three amino acids.

2. Elongation factors can provide additional energy for translation and can increase accuracy.

VI. Termination

1. The end of translation is signaled by stop codons

a. UAA, UAG,UGA

b. proteins, not tRNA, called release factors bind a stop codon in the A site and. The binding of these proteins forces the peptidyl transferase activity of the ribosome to add a water. As a result the ribosome disassembles and the protein is released.