RNA Processing and Translation

RNA Processing

Increases mRNA stability to facilitate export to cytosol.

Structure of 5’ cap of mRNA

• Occurs on all RNA transcripts destined to become mRNA

• 5’ to 5’ linkage of 7-methyl guanosine to mRNA

3 Capping Process

1. RNA Triphosphatase: Phosphate group cleaved at 5’ end of nascent mRNA.

2. mRNA Guanyltransferase: GTP hydrolyzed to GMP.  Joined the terminal phosphate group.

3. Guanin-N7-Methyltransferase: Methylation of GMP.

The Spliceosome

• Composed of small nuclear RNAs (snRNA) and proteins.

• Carries out intron splicing in 5 steps.

mRNA Splicing

1. U1 binds to 5’ splice site, U2 binds to 3’ splice site.

2. U6 stabilizes complex and replaces U1.

3. ATP Hydrolysis forms an active site between U2 and U6.

4. Intron is spliced out, exon is joined.

5. Exon junction complex marks splice site, lariat is degraded.

Lariat Structure

• Branch point adenine in the intron sequence attacks the 5’ splice site.

• The cut 5’ end becomes covalently linked to the 2’-OH of the ribose of the adenine.

• The free 3’-OH end of the exon then reacts with the start of the next exon sequence.

• The intron is released as a lariat structure that gets degraded.

Polyadenylation

• Occurs on all RNa transcripts that are destined to become mRNA.

• Added to the 3’ end of RNA.

• Protects RNA, helps with movement in the cytosol, and helps translation.

• Poly (A) Polymerase adds adenines using ATP hydrolysis.

𝛂-Trimyosin Splicing:

A protein that regulates mucle cell contraction.

mRNA export to cytoplasm

• The 5’ Cap and poly A tail are marked by recognition proteins.

• A group of proteins called the exon junction complex (EJC) is deposited onto the mRNA after successful RNA splicing has occurred.

Genetic Code

• 3 consecutive RNA nucleotides is called a codon and specifies 1 amino acid.

• 20 amino acids, some with >1 codon.

• Codons are written with the 5’-Terminal nucleotide to the left.

• AUG is the start codon.

• UAA, UAG, UAA are stop codons.

Reading Frames for RNA

• An open reading frame (ORF) begins at the start codon and ends at the stop codon.

• No codons overlap.

tRNA

tRNA has an anticodon that is complementary to the codon on mRNA and an amino acid attached to the 3’ end.

Translating the Genetic Code

• Aminoacyl-tRNA synthetase couples a particular amino acid to its corresponding tRNA through a process called charging.

• A tRNA coupled to its amino acid is called a charged tRNA.

Wobble

• The wobble effect refers to flexibility to tRNA in the 3rd nucleotide position.

Ribosome Structure

• Where translation takes place.

• Composed of a small and large subunit.

The Central Chamber

• Aminoacyl or A site.

• Peptidyl or P site.

• Exit or E site.

Bacterial Initiator tRNA

• Non-formylmethionine tRNA (fMet-tRNA)

• Only used for translation initiation.

Shine-Dalgarno Sequence

• Conserved sequence upstream of start codon in bacteria.

• Puts AUG in P site.

Translation Cycle

1. A tRNA carrying an amino acid binds to the vacant A site of the ribosome.

2. The carboxyl end of the polypeptide is joined by a peptide bond to the amino acid linked to the tRNA at the A-site.

3. A shift of the large subunit relative to the small subunit moves the two tRNAs into E and P sites of the large subunit.

4. The small subunit moves back to its original position relative to the large subunit.

• The cycle starts over to continue producing a polypeptide chain.

Translation Initiation in Eukaryotes

• The initiator tRNA, coupled to methionine, is loaded into a small ribosomal subunit along with translation initiation factors.

• Only the charged initiator tRNA can bind tightly to the P-site of a small ribosomal subunit.

• The loaded ribosomal subunit binds to the 5’ end of an mRNA molecule.

• The small subunit then moves forward. (5’ to 3’) along the mRNA searching for AUG.  When AUG is found, several initiation factors dissociate to make room for the large subunit.

• The binding of a release factor to an A-site bearing a stop codon terminates translation.

• Release factors bind to a stop codon that reaches the A-site on the ribosome which alters peptidyl transferase activity causing it to catalyze the addition of a water instead of an amino acid to the peptidyl tRNA.

• This reaction frees the carboxyl end of the polypeptide chain from its attachment to a tRNA molecule and the polypeptide is released.

• The ribosome releases the mRNA and dissociates into 2 separate units which can assemble on a new mRNA molecule.

Polyribosomes

• A series of ribosomes can simultaneously translate the same mRNA molecule.

• This allows multiple copies of a polypeptide to be made all at once, generating many proteins for the cell to use.

Protein Production in Eukaryotes

• The final concentration of each protein depends on the efficiency of each step.

• After a protein has been translated, its concentration can be regulated by degradation and its activity regulated by the binding of small molecules and the other post translational modifications.