Translation Apr 25, 2018

1968 Nobel Prize

Components of Translation 1. mRNA 2. tRNAs 3. Aminoacyl-tRNA synthetases 4. Ribosome In rapidly growing bacterial cells, 80% of the cells energy is used for translation 50% of the cells dry weight is proteins used for protein synthesis

mRNA Structure Bacterial mRNA and Eukaryotic mRNA Look at these pictures and compare and contrast bacterial and eukaryotic mRNA. a) Bacterial mRNA 5’ UTR ORF 3’ UTR In rapidly growing bacterial cells, 80% of the cells energy is used for translation 50% of the cells dry weight is proteins used for protein synthesis b) Eukaryotic mRNA 5’ UTR ORF 3’ UTR

mRNA Structure Bacterial mRNA and Eukaryotic mRNA Both have 5’ and 3’ UTRs (UnTranslated Region) Both have the start codon AUG Both have the stop codons UAG, UAA and UGA Both have coding sequences or ORFs (Open Reading Frames) a) Bacterial mRNA 5’ UTR ORF 3’ UTR In rapidly growing bacterial cells, 80% of the cells energy is used for translation 50% of the cells dry weight is proteins used for protein synthesis b) Eukaryotic mRNA 5’ UTR ORF 3’ UTR

mRNA Structure Bacterial mRNA only Ribosome-binding sites; also called Shine-Dalgarno sequence Eukaryotic mRNA only 5’ Cap (methylated guanine) Poly A tail Kozak Sequence (sometimes present); enhances ribosome binding a) Bacterial mRNA 5’ UTR ORF 3’ UTR In rapidly growing bacterial cells, 80% of the cells energy is used for translation 50% of the cells dry weight is proteins used for protein synthesis b) Eukaryotic mRNA 5’ UTR ORF 3’ UTR

Aminoacyl-tRNAs Synthetase Each of the 20 amino acids are attached to the appropriate tRNA by a dedicated aminoacyl-tRNA synthetase. So there are usually 20 aminoacyl-tRNA synthetase. “Charging” : couples a particular amino acid to its corresponding tRNA. In rapidly growing bacterial cells, 80% of the cells energy is used for translation 50% of the cells dry weight is proteins used for protein synthesis How is ATP used?

https://www.youtube.com/watch?v=W1eQNmtCCkw

Ribosomes The ribosome catalyzes a single reaction: The formation of a peptide bond. High Energy Bond Broken

RNA component of the Ribosome The RNA component in the ribosome acts as a ribozyme! It catalyzes the peptidyl transferase reaction which forms peptide bonds between two adjacent amino acids Prokaryotes The 23S RNA component in the 50S ribosome subunit Eukaryotes The 28S RNA component in the 60S ribosome subunit.

Peptidyl Transferase Reaction Peptidyl-tRNA = a tRNA molecule that is attached to an amino acid at its 3’ end. This amino acid is also part of the growing polypeptide chain Aminoacyl-tRNA = a tRNA molecule that is attached to an amino acid at its 3’ end. The amino acid is not yet incorporated into the polypeptide chain.

tRNA binding sites E= Exit site P= Binding site for the Peptidyl-tRNA (charged tRNA in which amino acid is attached to the growing polypeptide chain) A = Binding site for Aminoacylated-tRNA (charged tRNA in which the amino is NOT attached to the growing polypeptide chain).

Prokaryotic Initiation The start codon is typically AUG Therefore, the initiator tRNA anticodon is CAU Corresponding amino acid is methionine The initiator tRNA molecules is like no other The methionine has a special modification converting it to N-formyl methionine (fMet)

Prokaryotic Initiation Initiation factors bind to the 30S ribosome Recruits initiator tRNA (fMet) Ribosome with tRNA (fMet) binds to the ribosome binding site Large ribosomal subunit 50S binds to create the whole complex forming the 70S initiation complex

Eukaryotic Initiation Kozak sequence just upstream of the start codon. Increases the efficiency. 5’ Cap recruits the ribosome, the ribsome then scans the mRNA until it reaches a start codon 5’ UTR ORF 3’ UTR In rapidly growing bacterial cells, 80% of the cells energy is used for translation 50% of the cells dry weight is proteins used for protein synthesis

Translation Termination There are no tRNAs that recognize stop codons. A occupied “P” site but an unoccupied “A” is a signal to a protein called the release factor to binding the the A site and dissociate the ribosome assembly. In the standard genetic code, there are three mRNA stop codons: UAG ("amber"), UAA ("ochre"), and UGA ("opal" or "umber").