PROTEIN SYNTHESIS PROTEIN SYNTHESIS

How your cell makes very important proteins The production (synthesis) of proteins. 3 phases: 1.Transcription 2.RNA processing 3.Translation DNA  RNA  Protein

Nuclear membrane Transcription RNA Processing Translation DNA Pre-mRNA mRNA Ribosome Protein Eukaryotic Cell

RNADNA RNA differs from DNA RNAsugar ribose 1.RNA has a sugar ribose DNAsugar deoxyribose DNA has a sugar deoxyribose RNAuracil (U) 2.RNA contains uracil (U) DNAthymine (T) DNA has thymine (T) RNAsingle-stranded 3.RNA molecule is single-stranded DNAdouble-stranded DNA is double-stranded

1. Transcription DNA strands RNA Then moves along one of the DNA strands and links RNA nucleotides together. Nuclear membrane Transcription RNA Processing Translation DNA Pre-mRNA mRNA Ribosome Protein Eukaryotic Cell

2. RNA Processing Nuclear membrane Transcription RNA Processing Translation DNA Pre-mRNA mRNA Ribosome Protein Eukaryotic Cell

2. RNA Processing Intronsexons Introns are pulled out and exons come together. mature RNA molecule nucleuscytoplasm. End product is a mature RNA molecule that leaves the nucleus to the cytoplasm. Introns bad…… Exons good! Introns bad…… Exons good!

2. RNA Processing pre-RNA molecule intron exon Mature RNA molecule exon intron splicesome

3. Translation - making proteins Nuclear membrane Transcription RNA Processing Translation DNA Pre-mRNA mRNA Ribosome Protein Eukaryotic Cell

Eu- and Prokaryotic Ribosomes Eukaryotic cytoplasm Prokaryotes, Eukaryotic organelles (mitochondria, chloroplasts) (mitochondria, chloroplasts)

E: Exit site for free tRNA P: peptidyl-tRNA A: aminoacyl-tRNA E, P and A Sites of Ribosomes

Translation Translation Three parts: 1.Initiation: start codon (AUG) 2.Elongation: 3.Termination: stop codon (UAG)

The Initiation of Translation in Pro- and Eukaryotic Cells 5’ cap: After nucleotides have been synthesized, the 5’-end of the mRNA is capped 5’ to 5’ with a guanine nucleotide. Essential for the ribosome to bind to the 5’ end of the mRNA. Poly (A) tail: adenine nucleotides are added to 3’ end of mRNA. Stabilizes the mRNA, and plays an important role in transcription termination.

Initiation of Translation in Prokaryotes 50S 30S 50S 30S + RRF +3IF mRNA (fMet-tRNA f Met ) IF-2  GTP  fMet-tRNA f met 3 12 GTPfMet 30S Initiation Complex IF-1: 71aa, assists IF-2 binding IF-2: 890aa, binds initiator tRNA and GTP and GTP IF-3: 180aa, releases mRNA and tRNA from recycled 30S tRNA from recycled 30S subunit and aids (new) subunit and aids (new) mRNA binding mRNA binding RRF: ribosome release factor IF-2 = initiation factor 2 In complex with GTP, it brings fMet-tRNA f Met to the partial P site on the small subunit. Activates a GTPase activity in the small subunit, which allows dissociation of IF2, IF3, and IF1. mRNA 2GTPfMet

3 12 GTPfMet 30S Initiation Complex GTPfMet2 + GDP +Pi + GDP +PifMet 70S Initiation Complex fMet aa aa A site P site Elongation Phase of Translation

Simple process – involves only initiation factors (IFs) IF-1, IF-2 and IF-3 plus….. fMet-tRNA f Met and mRNA mRNA binds to small ribosomal subunit such that initiator AUG is positioned in the precursor to the P site In eubacteria, such as E. coli, the positioning of the initiator AUG is mediated by base pairing between the ribosome-binding site in the (5’) untranslated region of the mRNA and the 3’ end of the 16S rRNA Initiation of Translation in Prokaryotes

No involvement of mRNA 5’ end Shine – Dalgarno sequences +AUG initiation codons can occur within 5’ non-translated regions, and, may also occur within site(s) internal to the mRNA …….

Initiation of Translation in Prokaryotes

Initiation of Translation in Eukaryotes major differences to prokaryotic mRNA…… eukaryotic mRNAs possess a different 5’ ‘cap’ structure eukaryotic mRNAs are polyadenylated Bases around the initiating AUG influence the efficiency of initiation: RNNNAUGG (‘Kozak consensus’ sequence) Eukaryotic initiation factor eIF4 scans along mRNA from cap to find initiator AUG

(initiating) AUG open reading frame A(n) Stop codon 5’ ‘cap’ me7’ Gppp 43S ‘Scanning’ Model of Eukaryotic Initiation of Translation

(initiating) AUG open reading frameA(n) Stop codon 5’ ‘cap’ me7’ Gppp ‘scans’

(initiating) AUG AUG A(n) Stop codon 5’ ‘cap’ me7’ Gppp

(initiating) AUG AUG A(n) Stop codon 5’ ‘cap’ me7’ Gppp 60S

(initiating) AUG AUG A(n) Stop codon 5’ ‘cap’ me7’ Gppp Elongation Phase

Messenger RNA structure AAAAAAAAAAAAAAAAm7Gppp 5 ’ NCR AUG 3 ’ NCR stop Open reading frame ‘Cap’ Exon / exon splice boundaries Poly(A) tail

m7Gppp Messenger RNA structure – 5 ’ NCR AUG RNA stem-loop structures

Messenger RNA structure – 3 ’ NCR mRNA localisation elements (usually located in the 3’NCR) - binding sites for proteins which bind to the cytoskeleton - binding sites for proteins (located at specific cellular sites) which anchor the mRNA in that location AAAAAAAAAAAAA orf stop A / U rich Elements (AREs) - binding sites for stabilising / destabilising proteins