Translation is the process where the codons of mRNA are used to produce protein 11/26 What are the basics of translation? What are the 64 codons? Why only 61 tRNAs? How do eukaryotic and prokaryotic mRNAs differ? How does eukaryotic and prokaryotic translation differ? What are ribosomes composed of? What are the 4 ribosomal binding sites? How is tRNA “charged” with an amino acid? How is translation “initiated” What sets the “reading frame” of the codons in mRNA? How does peptide chain “elongation” occur? What “terminates” mRNA translation?

Transcription Review: how did we make mRNA Transcription Review: how did we make mRNA? The DNA duplex is unwound by RNAP to create access to the template strand for transcription into a complementary RNA sequence. NTPs are ONLY added (elongation) to the RNA 3’ end

Translation is the process whereby the mRNA sequence is converted into a protein sequence. Eukaryotic and Prokaryotic requirements are very similar! 1) Ribosome: Large subunit and Small subunit (Svedberg Units) 2) tRNAs are required to carry the 20 different amino acids 3) mRNA must have a start sequence (AUG) and a stop codon mRNA characteristics: Euk: mRNA is modified with a 5’-CH3 cap and 3’polyA-tail Prok: mRNA has a special ribosomal binding site just upstream of AUG Shine Delgano Sequence critical for initiation to occur. Three Main Processes to Translation of mRNA: 1) Initiation: mRNA Bound by ribosome, then tRNA binds AUG 2) Elongation: amino acids brought to new peptide by tRNAs 3) Termination: peptide and ribosome released from mRNA

The first 5’A-U-G-3’ encountered after the 5’ end of the mRNA is the start “codon”. Each base triplet after this is a specific “codon”. There are 20 standard amino acids There are 4X4X4=64 codons Base triplet is a codon 61 codons are for amino acids 3 codons terminate translation AUG codes for methionine AUG Codes for “Start” Termination occurs with: UAA UAG and UGA FrameShift Mutations change protein sequence by changing codon triplet identities on mRNA template CUGAUGA Add “A” CAUGAUGA  new protein Position if 3rd nucleotide is the most variable “Wobble”

Ribosomes are massive assemblies of proteins and pieces of rRNA that form discrete “small” and “large” subunits. Prokaryotes:50S+30S=70S Eukaryotes:60S+40S=80S Ribosomes work free in the cytosol or when membrane bound i.e. Rough Endoplasmic Reticulum (RER) Small ribosomal subunit bind mRNA first, then Large subunit Complex has four RNA-lined binding sites 1) mRNA-Site 2) A-Site (aminoacyl-tRNA) 3) P-Site (peptidyl-tRNA 4) E-Site empty tRNA exits from here! We call a ribosome a “ribozyme” because it has enzyme-like activity but it is made of RNA at its active site, not protein! The Proteins associated with it help regulate its shape and activity but are not entirely critical for its activity

The four binding sites on the ribosome are located deep inside the complex and the sites straddle the small and large subunits. Ribosomes consists of 2/3 rRNA and 1/3 Protein by weight.

tRNAs must be “charged” or “activated” by having their specific amino acid added to the hydroxyl located on their 3’ end by a special enzyme called aminoacyl-tRNA synthetase before translation can progress. Some amino acids have several different codons There are 20 different amino acids Each amino acid type has a specific aminoacyl-tRNA synthetase that recognizes all codons associate with that specific amino acid There are 20 different kinds of aminoacyl-tRNA synthetase AA+ATP+tRNAaminoacyl-tRNA(charged)+AMP+ 2 Pi “Charging costs two Pi-Pi bonds! Expensive tRNA Anticodon binds complementary mRNA Codons mRNA ‘5-AUG-3’ binds ‘3-UAC-5’ of tRNA RNA=RNA binding is not as stable as DNA Double helix “Wobble” refers to the fact that the bases in the codon (especially the third) can be variable.

How is the translation of mRNA to protein initiated? Four Steps to Initiation: 1) Small ribosomal subunit binds 5’ end of mRNA 2) met-tRNA binds ribosome and crawls along mRNA until an AUG is encountered 3) Small subunit binds AUG and then attracts the large ribosomal subunit 4) Large subunit binds to small unit/mRNA to complete the ribosomal complex Initiation is now completed! Note: The first AUG encountered by the ribosome sets the “Reading Frame” for all base-triplets (codons) that come after it, mRNA is read 3 bases at a time. A single mRNA can have several ribosomes on it at one time In prokaryotes a Shine-Delgano Sequence of –AGGA- must precede the AUG for initiation (not required in eukaryotes) Prokaryotic Shine-Delgano sequence on mRNA ‘5-AGGA-3’ binds ‘3-UCCA-5’ on the complementary rRNA stabilizing initiation complex. In prokaryotes the first amino acid is always formyl-methionine fMet => Methionine with a formyl group on its amino end to make it non-reactive while being created

Elongation: additional charged tRNAs bring additional amino acids to the open A-site on the ribosome. The new aminoacyl-tRNA accepts the old peptide chain. It becomes the P-site and the old tRNA shifts into the E-site. Peptide elongation process uses a “ribozyme” called peptidyltransferase.

Termination of translation occurs when a stop codon UAA, UAG or UGA is exposed at the A-site. Stop codon causes a release factor (protein) to bind this open site causing the nascent peptide to be released from the 3’-end of tRNA at the P-site. After release, the ribosome and mRNA also dissociate.

Start Codon: AUG Termination Codons: UAC UAG and AGA 1) Given this mRNA how many amino acids long is the protein? 5’UUCGAUG-GCC-UCU-UGC-AUG-GCG-UAG-UUU-AG…3’ 1 5 10 30 2) If Base #5 (A) is removed a new reading frame is established, where does synthesis start? 5’UUCGUGGCCUCUUGC-AUG-GCG-UAG-UUU-AG….3’ 1 5 10 go 29 3) If an extra base (G) is ADDED immediately after #7 the total mRNA becomes 31 bases, how many amino acids are produced? 5’UUCGAUG-GGC-CUC-UUG-CAU-GGC-GUA-GUU-UGC...3’ 5go 10 31