Protein Synthesis AY24-25 PDF

Molecules of Life: Protein Synthesis Dr Sarah J. Jones [email protected]  Revision: Eucaryotic mRNA AUG CODON START STOP In more detail: 2 Revision: tRNA Structure The ant...

Molecules of Life: Protein Synthesis Dr Sarah J. Jones [email protected]  Revision: Eucaryotic mRNA AUG CODON START STOP In more detail: 2 Revision: tRNA Structure The anti-codon loop is the most important part of tRNA. It recognises the codon on the mRNA indicating the amino acid required. The anti-codon loop is a 3 nucleotide sequence which base-pairs with the codon on the mRNA. The amino acid is covalently attached at the acceptor stem by an amino acylation reaction catalysed by the enzyme: aminoacyl tRNA synthetase 3  Translation: codon-anticodon basepairing Met tRNA anticodon During translation, anticodons of tRNA molecules form base pairs with codons on mRNA Codons and anticodons each consist of 3 nucleotides Therefore, the genetic code is based on triplets mRNA is decoded in a 5’ to 3’ direction, one codon at a time Protein is synthesised from amino terminus (NH2)  carboxy terminus (COO-) 4  The Genetic Code Start codon Stop codons Degenerate: many amino acids have more than one codon (also called REDUNDANCY) Unambiguous: each codon codes for only one amino acid (or a Stop) 5 Synthesis of Aminoacyl tRNAs by Amino Acyl Synthetase 2 STEPS: Each amino acid is activated Then linked to a specific tRNA by a high energy bond Each amino acid has at least one specific tRNA and a corresponding synthetase. The synthetase can prevent attachment of the incorrect amino acid to the tRNA. This is vital to the fidelity of the translation 6  Amino Acyl Synthetase STEP 1: Amino acid + ATP -> Aminoacyl-AMP + PPi STEP 2: Aminoacyl-AMP + tRNA -> Aminoacyl-tRNA + AMP OVERALL REACTION: Amino acid + ATP + tRNA -> Aminoacyl-tRNA + 2Pi In eucaryotes, there is a separate synthetase for each amino acid, which functions for all the tRNA that carry that amino acid. The synthetase can check to see if the correct amino acid is attached to the tRNA. 7  Translation has 3 Phases: 1. Initiation of protein synthesis 2. Elongation of protein chain and translocation of the ribosome 3. Termination There are specific sets of protein factors for each phase 8  Components of Translation 1. Amino acids 2. tRNAs 3. Aminoacyl-tRNA synthetases 4. A specific set of protein factors for each step: Initiation of protein synthesis Elongation of polypeptide chain and translocation Termination 5. ATP and GTP as sources of energy 6. Ribosomes 7. mRNA ATP = adenosine triphosphate GTP = guanosine triphosphate 9  Revision: Ribosomes Ribosomes are composed of proteins and RNA: Procaryotic ribosomes contain three rRNA molecules and 50+ proteins Eucaryotic ribosomes contain four rRNA molecules and 80+ proteins INTACT CELL TYPE rRNA SUBUNIT SIZE RIBOSOME 23S, 5S large 50S Procaryotic 70S 16S small 30S 28S, 5.8S, 5S large 60S Eucaryotic 80S 18S small 40S S=Svedberg units – sedimentation constant depending on mass, density & shape 10 Just for reference: Svedberg (S) Values Subunits of ribosomes are often described by their Svedberg (s) values, based upon their rate of sedimentation in a centrifuge. Eucaryotic ribosomes have a Svedberg value of 80S and are comprised of 40S and 60S subunits. Prokaryotic cells contain 70S ribosomes, each comprised of one 30S and one 50S subunit. Svedberg units are not additive, so the values of the two subunits of a ribosome do not add up to the Svedberg value of the entire organelle. This is because the rate of sedimentation of a molecule depends upon its size and shape, rather than simply its molecular weight. Do not learn! 11 Procaryotic Ribosome 50S subunit: Protein (pink/red) 23S RNA (yellow) 5S RNA (orange) 30S subunit: Protein (blue) 16S RNA (green) Figure from Biochemistry by Berg, Tymoczko and Stryer, 7th edition 2012 12  Ribosomes have 3 tRNA Binding Sites A = Aminoacyl The binding site for the charged tRNA anticodon which binds to the mRNA codon P = Peptidyl The binding site for the tRNA that carries the growing polypeptide chain E = Exit The binding site for an uncharged tRNA about to be released and recycled Each tRNA is in contact with the 30s and 50s ribosomal subunits tRNA at sites A and P are base-paired with mRNA Figure from Biochemistry by Berg, Tymoczko and Stryer, 7th edition 2012, also 5th edition 2002. 13 Initiation of Translation: In procaryotes At least 8 components are required to form an INITIATION COMPLEX, including: mRNA, 30s ribosomal subunit, *fmet tRNA, GTP and 3 initiation factors (proteins) 16s RNA of the 30s ribosomal subunit binds to a specific mRNA sequence (Shine-Dalgarno) near the start codon, AUG which codes for Methionine Once the 30s initiation complex is complete, the 50s subunit binds, hydrolysis of GTP to GDP occurs and initiation factors are released *A modified form of methionine (met) is carried by the first tRNA, N-formylmethionine (fmet) Finding the Protein-Coding Region 14 Initiation: Eucaryotes Finding the Protein-Coding Region 40s ribosomal subunit forms a complex with 4 initiation factors and Met tRNA, which has GTP and another initiation factor bound to it Factors bound to the mRNA 5’ cap direct the preinitiation complex to it. The complex then scans the mRNA and translocates to the start codon (AUG) using energy from ATP hydrolysis The 60s ribosomal subunit then binds to the 40s complex Met tRNA is directed to the P site hydrolysing GTP and releasing initiation factors 15 Don’t worry about the names of the individual initiation factors (IF) ! Translocation & Peptide Bond Formation 1. Peptidyl transferase (a ribozyme) catalyses peptide bond formation between amino acids in the P and A sites and breaks the bond between tRNA and its amino acid in the P site. 2. The peptide is now transiently bound to the A site. 3. A elongation factor (eEF-2) moves the ribosome along the mRNA by one codon (GTP hydrolysed) 4. ‘Empty’ tRNA is now in the E site, can exit and become reloaded with another amino acid 5. tRNA with the growing peptide is now in the P site 6. A site is free for the next aminoacyl-tRNA Ribozyme = RNA with catalytic activity 16  Termination Occurs when the A site of the ribosome encounters a stop codon on the mRNA e.g. UAA, UAG or UGA No charged tRNA base-pairs with stop codons Release factor (eRF) binds the stop codon, GTP is hydrolysed The finished protein is cleaved off The components, rRNA, mRNA and tRNA dissociate Process can then start again with small 40s subunit being bound by initiation factors ready for translation of a new protein 17 Revision: Antibiotics that Target Protein Synthesis Antibiotic Target Tetracycline Bacterial ribosome A site Streptomycin Bacterial 30s ribosomal subunit Erythromycin Bacterial 50s ribosomal subunit Chloramphenicol Bacterial ribosome peptidyl transferase Puromycin Causes premature termination *Cycloheximide Eucaryotic 80s ribosome - translocation *Toxic to humans, used as a fungicide, was used in agriculture, generally used only for research purposes now. 18  Translation – Reading Frames Theoretically its possible to translate the mRNA molecule in three different reading frames – depends on where translation starts 19  Types of Mutations Insertion or deletion -> frameshift mutation, causing a shift in reading frame Altered base -> point mutation, maybe a silent mutation 20  Effects of Mutations Point Mutations (changes in a single base in the DNA): 1. Missense Mutation: Results in a change of amino acid primary sequence Can change protein function, e.g. altered haemoglobin in sickle cell anaemia 2. Nonsense Mutation: Creates a new STOP codon Changes the length of the protein due to premature stop of translation 3. Silent Mutation: No change in amino acid sequence Due to degeneracy of the genetic code (each amino acid has more than 1 codon) No effect on protein function OTHER TYPES OF MUTATION: Frameshift Mutation: Insertion or deletion of a single base (or two) Changes reading frame of translation into protein, can cause premature stop of translation 21 Examples: Cystic fibrosis – many mutations of the CFTR gene have been described, the most common is a deletion which leads to loss of the amino acid, Phenylalanine. This results in a protein which does not function correctly. (CFTR = cystic fibrosis transmembrane conductance regulator) http://scibritts.wordpress.com/2011/02/25/the-characteristics-of-cystic-fibrosis/ Sickle cell anaemia – just one base in the DNA is changed GAG -> GTG (Glutamate -> Valine). The haemoglobin (HbS) behaves normally until exposed to low oxygen tensions where large fibrous aggregates form, distorting the erythrocytes and affecting blood flow. 22 Examples Related to Tooth Development: MSX1 and MSX2, are genes encoding transcription factors involved in the initiation, developmental position (patterning) and differentiation of tooth buds. PAX9 is another transcription factor involved in the early stages of tooth development. Mutation of genes PAX9 and MSX1 have been identified as the main causes of hypodontia (1-6 missing teeth, excluding 3rd molars) and oligodontia (>6 missing teeth, excluding 3rd molars). Mutation of MSX1 is associated with missing premolars and certain syndromic conditions. AXIN2 mutation is associated with familial oligodontia and anodontia; the affected person also has a higher susceptibility to colorectal cancer. 23  What Happens to the Finished Protein? Targeting moving a protein to its final cellular destination there are many possible locations within a cell depends on the presence of specific amino acid sequences within the translated protein Modification addition of further functional chemical groups Degradation unwanted or damaged proteins have to be removed 24  Protein Folding For a newly synthesised protein to be biologically active it must be correctly folded i.e. have the correct 3D shape Proteins achieve this with the help of molecular chaperones Chaperones promote correct folding, assembly and organisation of proteins and macromolecular structures Examples of chaperones include heat shock proteins (HSP60, HSP70) and protein disulphide isomerase 25  Free and Bound Ribosomes Free ribosomes in the cytosol make proteins destined for: - cytosol - nucleus - mitochondria - translocation post-translationally Bound ribosomes on the rough endoplasmic reticulum (RER) make proteins destined for: - plasma membrane - endoplasmic reticulum - Golgi apparatus - secretion - translocation co-translationally 26  Protein Targeting The cellular fate of proteins is determined by their signal peptide sequence Proteins targeted for secretion, membranes or cellular organelles have a 20-30 amino acid sequence at their N-terminus called a signal sequence Shortly after translation, the signal sequence is recognised by a ribonucleoprotein complex called the Signal Recognition Particle (SRP) SRP binds to the sequence and stops the translation The complex is then targeted to the signal recognition particle receptor (SRPR) on the endoplasmic reticulum The signal sequence is then inserted through the membrane, SRP dissociates and translation continues, the protein can then be transferred to the Golgi apparatus and on to its destination. The signal sequence may or may not be removed. 27 28  Post-translational Modifications Glycosylation = addition and processing of carbohydrates in the ER and the Golgi Other examples: Formation of disulfide bonds in the ER Folding and assembly of multi subunit proteins in the ER Specific proteolytic cleavage in the ER, Golgi, and secretory vesicles 29