FUNBIO 13 2024 Protein Synthesis PDF

25/10/2024 Translation: the prokaryotic ribosome, and protein synthesis Class Foundation Year Course Fundamentals of Human Biology Code FUNBIO.13 Lecturer Prof Michael B. Keogh 1 Learning Objectives ALO 1 Describe t...

25/10/2024 Translation: the prokaryotic ribosome, and protein synthesis Class Foundation Year Course Fundamentals of Human Biology Code FUNBIO.13 Lecturer Prof Michael B. Keogh 1 Learning Objectives ALO 1 Describe the molecular basis of the ribosome & subunits structure. ALO 2 Discuss ribosomal function differentiating between bound and free populations ALO 3 Describe the ribosomal structure and describe the events occurring at specific sites on the ribosome during the translation process; initiation, elongation, translocation and termination. ALO 4 Discuss the role of the primary molecules involved in the translation of RNA to protein; RNA template, ribosomal structure, and transfer RNAs. ALO 5 Describe the steps involved in the process of RNA translation ALO 6 Classify post-translational modification of proteins and their role 2 1  25/10/2024 ALO 1&2 The Ribosome (E. coli) ~15,000 per cell; equivalent to ~33% of cell mass Composed of a collection of many different proteins and ribosomal RNAs, organised into two sub-units –Large (50S) –Small (30S) The small subunit fits into the large subunit rRNA is single stranded but there is extensive intermolecular base-pairing forming complex folded structures Unlike mRNA and tRNA, rRNA is catalytic and does not transfer information 3 ALO 1&2 The Ribosome (E. coli) Ribosomes are classified as being Large Subunit Small Subunit 50S: 30S: either "free" or "membrane- 5S rRNA 16S rRNA bound” 23S rRNA 21 Proteins 34 Proteins 4 2  25/10/2024 ALO 3 The Ribosome (E. coli) Ribosomes have 3 pockets in the general structure 1. A-site 2. P-site A site – Aminoacyl site 3. E-site The attachment site for the aminoacyl tRNA P site – Peptidyl site The site holding the growing peptide chain E site – Exit site Where tRNAs that have delivered their amino acid exit the chain 5 ALO 4 Transfer RNAs Single-stranded molecules typically 70-80 nucleotides in length which fold extensively due to intra-molecular base-pairing Functional units: 1. Anticodon site for binding to mRNA 2. Recognition site for aminoacyl-tRNA synthase where complementary amino acid will be added 3. Attachment site for amino-acid Fold to give an L-shaped structure 4. Recognition site for ribosomes The amino-acid is added to the 3’ end of the tRNA molecule to give an aminoacyl-tRNA The “anti-codon loop” mediates recognition of codons within the mRNA Function: Link between mRNA code and amino acid sequence 6 3  25/10/2024 ALO 4 Amino-acyl tRNAs Activated precursors for protein synthesis Also called ‘charged tRNAs’ Each amino-acid is coupled to its specific “carrier” tRNA molecule by a two-stage reaction, which requires energy in the form of ATP 1)adenylation: Amino acid reacts with ATP and AMP is transferred to AAs 2) transfer of the amino acid to the 3’ of the tRNA and release of AMP Catalysed by specific Aminoacyl-tRNA Synthetase enzymes At least one Aminoacyl-tRNA Synthetase exists for each amino-acid type 7 ALO 4 mRNA – Ribosomes - tRNA The ribosome “reads” the mRNA in a 5’ to 3’ direction Translated in groups of three bases termed codons Ribosome moves along the mRNA (from upstream to downstream) until it locates the start codon – AUG Nascent protein grows from the N-terminus to the C- terminus Ribosome stops reading mRNA at stop codon – UAA, UAG, UGA 8 4  25/10/2024 ALO 5 mRNA translation into protein The ribosome “reads” the mRNA in a 5’ to 3’ direction Peptides/ nascent protein grows from the N-terminus to the C-terminus Translated in groups of three bases termed codons Ribosome moves along the mRNA until it locates the start codon – AUG (Therefore all proteins begin with the amino-acid Methionine (AUG) mRNA Ribosome stops reading mRNA 5’ 3’ at stop codon – UAA, UAG, UGA N C Polypeptide 9 ALO 5 Translation: Protein Synthesis Translation is the conversion of the mRNA code into an amino acid sequence Template: Start Stop tRNA: Ribosomes: 10 5  25/10/2024 ALO 5 Translation: Protein Synthesis Template: Start Stop 5’- AUG GAC CAG UUU AGG UCG CCU CAA AAG GCC AGC UGA -3’ Met – Asp – Gln – Phe – Lys – Ser – Pro – Gln – Lys – Ala – Ser UAA UAG UGA 11 ALO 5 Codons A triplet code Sequence of 3 bases, the ‘Codon’, specifies an amino-acid Three bases can code for 43 = 64 amino-acids The code contains: –61 amino-acid coding codons which code for 20 amino acids –3 Stop codons [UAA, UAG, UGA] –1 Start codon [AUG] which also encodes Met 12 6  25/10/2024 Translation/Protein Transcription/RNA Synthesis: Synthesis: Initiation, Elongation, & Termination GO UP! STOP ⇒⇒⇒⇒⇒ ⇒⇒⇒⇒⇒ Stages of translation 1.Initiation (Go) 2.Elongation (Up) 3.Termination (Stop) 13 Protein Synthesis: Initiation GO 1. Initiation a)Assembly of small ribosome subunit along the mRNA b)Binding of the first amino acid carried on its tRNA to the ribosome. c)then the large subunit around the beginning of the mRNA Binding of the first amino acid carried on its tRNA to the ribosome Initiator tRNA – fMet.tRNA N-Formylmethionine (specific to bacteria) Therefore all proteins begin with the amino-acid Methionine At initiation: A-site: Vacant P-site has fMet tRNA E-site: Vacant 14 7  25/10/2024 ALO 5 Protein Synthesis 2. Elongation a)Second aminoacyl tRNA enters the A-site of the ribosome a) Codon-anticodon recognition b)Peptidyl-transferase reaction a) Formation of peptide bond between initiator amino acid (P-site) and second amino acid (A-site) c)Uncharged tRNA enters E-site and leaves the ribosome d)The peptidyl-tRNA moves from the A-site to the P-site 15 ALO 5 Protein Synthesis The mRNA moves on by three bases. The A site is now aligned with the next codon in the mRNA and ready to accept the next tRNA molecule 16 8  25/10/2024 ALO 5 Prokaryotes Eukaryotes 17 ALO 5 Protein Synthesis: Elongation UP! 2. Elongation Second aminoacyl tRNA recognises the codon in the A site of the ribosome Codon-anticodon recognition Peptidyl-transferase reaction Formation of peptide bond between initiator amino acid (P-site) and second amino acid (A-site) Translocation 1.The uncharged (“empty”) tRNA leaves the P-site 2.The peptidyl-tRNA (now 2 amino-acids) moves from the A-site to the P-site 3.The mRNA moves on by three bases 4.The A site is now aligned with the next codon in the mRNA and ready to accept the next tRNA molecule 18 9  25/10/2024 ALO 5 Protein Synthesis: Termination STOP Recognised by Release Factors mRNA contains “Stop codons” (RF1,RF2, RF3) UAA, UGA & UAG Bind to the A-site These do not code for any amino acid Cause the peptide in the P-site to be cleaved from the tRNA and released 19 ALO 5 Translation & Polysomes Each ribosome synthesizes just one polypeptide at a time Many ribosomes can translate a single mRNA molecule Referred to as a “Polyribosome” or “Polysome” 20 10  25/10/2024 ALO 6 Post-translation Modifications - refers to the covalent and generally enzymatic modification of proteins following protein biosynthesis. After synthesis most proteins are modified further before they are fully functional Only 20 amino acids – cell uses post- translational modifications (over 200 types) to increase diversity, including: Disulphide bond formation (e.g. insulin) Proteolytic cleavage (e.g. insulin -> A and B chains) Addition of carbohydrate (Glycosylation) Addition of phosphate (Phosphorylation) Addition of lipid groups (Prenylation, Acylation) Hydroxylation (e.g. Collagen) 21 Post-translation Events ALO 6 Proteins must fold up into their natural and biologically-active conformation. Mostly this is spontaneous -chemical modification of a protein may occur after translation to extend the range of functions of the protein by attaching functional groups (e.g. Phosphorylation and Glycosylation) Some proteins need helper proteins known as “Chaperones” to assist in the folding process Recall FUNBIO 3 22 11