FFP1 Translation 2023-2024 PDF

Royal College of Surgeons in Ireland – Medical University of Bahrain JC2 Translation and Dynamic Molecular Medicine – MM6 (LT6) Regulation of proteins Prof. Raymond Stallings, Feb. 2012 Module : Foundations for Practice 1 FFP1 Class : MedYear 1 semester 1 Lecturer : Paul O’Farrell Date : 16...

Royal College of Surgeons in Ireland – Medical University of Bahrain JC2 Translation and Dynamic Molecular Medicine – MM6 (LT6) Regulation of proteins Prof. Raymond Stallings, Feb. 2012 Module : Foundations for Practice 1 FFP1 Class : MedYear 1 semester 1 Lecturer : Paul O’Farrell Date : 16 October 2023 Learning Objectives Describe the genetic code in the context of codons and how this code facilitates the translation of amino acids Describe the stages of translation - initiation, elongation, termination Discuss the importance of regulating protein levels Describe how phosphorylation can affect protein activity, function and localization Discuss the different forms of post-translational modification of proteins Information flow Transmission “The Central Dogma” of molecular biology m Other RNAs - Francis Crick 1956 Action Action Overview of Translation Information in mRNA is translated into amino acids (protein) 3 Stages: Initiation Elongation Termination Translation: protein synthesis Basic Requirements: Template mRNA Catalyst Ribosome (ribosomal proteins + rRNA) Activated precursors aminoacyl-tRNA tRNA molecules coupled to amino acids via energy-rich bonds Release/Termination factors The sequence of bases in the mRNA determines the sequence of amino acid residues in the translated protein  Any variation in the sequence of mRNA bases can affect the sequence of amino acids in the protein  such variation may result in differences in function of the protein The Genetic Code translates nucleic acid sequences to amino acid sequences The genetic code is the dictionary that translates DNA sequence in to amino acids But there are 20 amino acids and only 4 nucleotides.. What size is a codon (or “word)? If 1 nucleotide coded for amino acid – 4 possibilities 2 nucleotides, 4 X 4 = 16 possibilities 3 nucleotides, 4 X 4 X 4 = 64 possibilities Each “word” of DNA consists of 3 nucleotides/bases = 1 amino acid The Genetic code has been determined The genetic code is unpunctuated: reading frames Open Reading Frame (ORF): A set of codons that runs continuously, beginning with a start codon and ending with a stop codon The ‘reading frame’ is determined by the position of the start codon In protein synthesis, only one ORF contains useful information The genetic code is unpunctuated: reading frames Reading frame 1 AUGUUUAAAUGGUGA The dog ate the cat start/met Phe Lys Trp Stop Reading frame 2 AUG UUU AAA UGG UGA T hed oga tet hec at Cys Leu Asn Gly Reading frame 3 AUG UUU AAA UGG UGA Th edo gat eth eca t Val Stop Start Val insertions or deletions of nucleotides can cause “frame-shift” mutations The genetic code is specific, universal redundant and non-overlapping Specific – a specific codon always codes for the same amino acid Universal* – applies to all species. Conserved from early stage of evolution Redundant – a given amino acid can be coded for by several different codons Non-overlapping – the code is read from a fixed starting point, as a continuous sequence of bases, taken 3 at a time Changes in coding sequence can cause disease: Huntington’s disease Sometimes a codon can become amplified between generations (e.g. father carries 4 copies but he passes on 8 copies to his child Each extra copy of the codon = an extra copy of the amino acid in the protein. Lippencott’s biochemistry 31.4 This can lead to changes in protein structure, and sometimes accumulation and deposition of the protein in the cell Changes in coding sequence can cause disease : SCD missense mutation in the β-globin gene - single nucleotide substitution (A  T) in the codon for amino acid 6 converts a glutamic acid codon (GAG) to a valine codon (GTG) autosomal recessive https://thestrangeandspectacularworldofbiochemistry.wordpress.com/2013/03/24/protein-mutations-and-sickle-cell-anemia tRNA is an adaptor molecule that translates the genetic code Nucleic acids and proteins are very different Francis Crick proposed that there must be adaptor molecules: “I cannot conceive of any structure for either nucleic acid acting as a direct template for amino acids …..each amino acid would combine..with a small molecule which would combine specifically with the nucleic acid template….there would be 20 different kinds of adaptor molecule” Crick, FHC. 1958. Unpublished letter to the “RNA Tie Club” The adaptor molecule is transfer-RNA tRNA tRNA molecules fold to give an L-shaped structure Base-pairing between nucleotides allows the tRNA to fold up into a specific “secondary structure”1, which gives rise to a specific 3-dimensional shape2 tRNA molecules also undergo some post-transcriptional modification The amino-acid is added to the 3’ end of the tRNA molecule to give an aminoacyl-tRNA by Aminoacyl-tRNA-synthetase. Specificity is derived from the 3D structure of the particular tRNA* The “anti-codon loop” mediates recognition of codons within the mRNA by base-pairing ~ 80 nucleotides There are about 50 different tRNA molecules 1 “cloverleaf” secondary structure 2 L-shaped three-dimensional shape tRNA anticodons pair with mRNA codons following the Watson-Crick rules, generally UCC codes for Serine The 3rd base in a codon is known as the “wobble” position 61 aa coding codons and 50 tRNAs? : some tRNAs must match more than one codon The “Wobble” base is last base in codon (first in anticodon) Allows flexibility/efficiency in use of tRNA A single tRNA species carrying an AA can recognize more than one codon (serine is shown) The ribosome contains protein and RNA Ribosomes are the factories in which protein synthesis occurs. They are complexes of protein and rRNA rRNA - Extensive secondary structuring – similar to tRNA Ribosome brings tRNA and mRNA together to translate nucleotide sequence of mRNA into amino acid of a protein Structure of the ribosome A site: aminoacyl site, binds incoming aminoacyl tRNA P site: peptidyl site, tRNA in P site contains growing polypeptide chain E site: exit site, contains deacylated tRNA Decoding centre: ensures only tRNAs with anticodon that matches codon are accepted into a site Peptidyl transferase centre: where peptide bond formation is catalysed Initiation of Translation requires a) Assembly of components required for chain formation: 40s ribosomal subunit The mRNA to be translated The tRNA specified by the first codon in the mRNA (tRNAmet) GTP (which provides the required energy) Initiation factors (to help the ribosome recognise the sequence for the start of translation) b) Recognition of the start codon by the tRNAmet molecule c) Addition of 60s ribosomal subunit to complete assemble Initiation of Translation 1) Initial Assembly of components 2) Recognition of start codon.. 3) Final Assembly Note: initiator tRNA is in P site Elongation Elongation involves the addition of amino acids to the carboxyl end of the growing chain Ribosome moves along mRNA being translated - in 5’ to 3’ direction 1. Next required aminoacyl-tRNA is delivered to “A” site (with help of elongation factors) 2. Peptide bonds are formed between adjacent amino acids – facilitated by the enzyme peptidyltransferase 3. After a bond is formed the ribosome is translocated three nucleotides in 3’ direction - to the next codon. – Growing chain thus moved in to “P” site. – “Uncharged” tRNA moves to “E” (exit) site & released – “A” site free to accept next tRNA  Elongation initiation, P site contains initiator tRNA...... Elongation involves the addition of amino acids to the carboxyl end of the growing chain loading of correct (codon-anticodon matching) aminoacyl tRNA in A site peptidyl transferase activity of ribosome (peptide bond formation) – ( at this point the peptide chain is in A site, and there is a ‘empty’ tRNA in the P site) ribosome translocation (by one codon along the mRNA) peptidyl chain is now in P site A site ready to accept the next aminoacyl tRNA Empty tRNA moves into E (exit site) and is released Path through ribosome for each tRNA is A – P – E sites Elongation Step 1 Step 3 1. Next required aminoacyl- Step 2 tRNA is delivered to “A” site 2. Peptide bonds are formed 3. Growing amino acid chain Step 4 now in “A” site 4. After a bond is formed the ribosome is translocated 3 nucleotides in 3’ direction, to the next codon. Termination Occurs when one of three termination codons arrives in the “A” site Stop codon recognised by a release factor Release factor binds to “A” site – which causes: 1. The newly synthesised protein to be released. 2. Disassembly of the tRNA-ribosome- mRNA complex Note: ribosomal subunits, mRNA, tRNA and protein factors can be recycled & used to make additional proteins Regulation of Protein Activity As many key functions that occur in cells are dependant on the activities of proteins, regulating protein activity is essential The cell uses multiple ways of regulating protein activity Protein Levels Protein Location Post-translational modification Balancing Protein Synthesis with Degradation Synthesis Degradation Protein turnover is a normal process: “Housekeeping” proteins get damaged and need to be replaced Some proteins involved in key cellular processes need to be degraded to ensure tight control over their levels – eg Cyclins control the progression of a cell around the cell cycle Rapid turnover of some proteins is necessary to allow their levels to change How are proteins degraded? 1. Lysosomes Lysosomal degradation The Lysosome is a membrane-bound organelle that contains powerful degradative enzymes These enzymes will non-specifically degrade proteins, other molecules, organelles, microbes and other materials that get sent there. The enzymes only function at the low pH found inside the lysosome Proteasomal degradation Poly- Proteins can be labelled or ‘tagged’ Ubiquitin tag for degradation by : ‘polyubiquitination’ Once polyubiquitinated the target protein is recognised by Ubiquitin protein proteasome and degraded Proteasome: a multiprotein, hollow, barrel shaped complex containing protease activity, sequestered inside the barrell Found in the nucleus or cytoplasm Protein “caps” on the ends of the proteasome recognise tagged proteins and feed them in Polypeptide chains can be modified after translation Creates mature functional protein from immature protein Proteolytic cleavage Eg processing of insulin from pre-pro form Eg trypsinogen  trypsin Covalent modifications Disulfide bond formation Phosphorylation – often activates/inactivates; important control mechanism Glycosylation – often extracellular proteins Farnesylation, GPI – to target to membranes etc, Ligand binding Ligand Binding Changes shape of protein Active conformation Release of inhibitory subunits Change of cell location Steroid hormone receptors bind their ligand (i.e: steroid) Get conformational change Now dimerise and enter nucleus Proteolytic Cleavage Zymogens: inactive “pro-enzymes” Activated by cleavage enterokinase Proteolytic cascades: clotting Reversible Phosphorylation of proteins Phosphate group may be added to serine, threonine and tyrosine residues Very tightly regulated Kinases add phosphate; Phosphatases remove it Ratio of phosphorylated S:T:Y is approx 100:10:1 but Tyrosine phosphorylation has the most important effects Further resources: Reading: Lippencott’s Biochemistry (3rd ed) – chapter 31 – protein synthesis Strachan & Read. Human Molecular Genetics 3. Chapter 1 – DNA structure & gene expression The proteasome and ubiquitination (Nobel prize in Chemistry 2004 Phosphorylation (Nobel prize in Physiology or Medicine 1992) Viewing: Translation animation Post-translational modification