Lecture 3 Protein Structure, Function and Translation.pdf

Molecular Diagnostics Translation / Protein Synthesis Lecture 3 Building Blocks of Protein • Amino acids are the structural units (monomers) that make up proteins. • There are the 20 biologically active amino acids in humans. They are encoded directly by the codons of the universal genetic code ar...

Molecular Diagnostics Translation / Protein Synthesis Lecture 3 Building Blocks of Protein • Amino acids are the structural units (monomers) that make up proteins. • There are the 20 biologically active amino acids in humans. They are encoded directly by the codons of the universal genetic code are called standard or canonical amino acids • There are nine Essential amino acids (indispensable) amino acid , which cannot be synthesized de novo ), and have to get them from diet. – histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. • Amino Acids join together to form short polymer chains called peptides or longer chains called either polypeptides or proteins. • Basic Amino Acid Structure R Group Residues Peptide Bond Formation The Genetic Code • Codon – a sequence of three nucleotides that together form a unit of genetic code in a DNA or RNA molecule, and the genetic information is translated into proteins by living cells. • Nonoverlapping • Universal in plant and animal kingdoms (ex. Mitochondrial code) • Degenerate (wobble base codon) – The first two positions of the mRNA codon observe Watson-Crick base pairing rules (A-U, C-G) The third position exhibits wobble • Read by complementary tRNA linked to aa • Initiation codon = AUG (met) • Stop codons (UAA, UGA, UAG) The Genetic Code All 64 possible 3-letter combinations of the DNA coding units T, C, A and G (43) are used either to encode one of these amino acids or as one of the three stop codons that signals the end of a sequence. While DNA can be decoded unambiguously, it is not possible to predict a DNA sequence from its protein sequence. Because most amino acids have multiple codons, a number of possible DNA sequences might represent the same protein sequence. Translation • Information decoding • High energy consuming process – consumes 90% of cells energy – 4 ATP / aa Protein Synthesis Players • • • • Ribosome / rough ER / rER (rRNA + protein) tRNA = anticodon with aa mRNA = codon Ribosome has 2 sites which associate with mRNA – P site (initaition) – A site (elongation) Protein Synthesis Steps • Initiation – 1st aa always methionine (Met) at P site – Template = mRNA – mRNA moves down in register (A site) and codon is read by anticodon of tRNA • Elongation – new aa brought in to match new codons and peptide bonds formed • Termination – Stop codon (UGA,UAA,UAG) Massager RNA (mRNA) • Carries instructions from DNA to the rest of the ribosome. • Tells the ribosome what kind of protein to make Transfer RNA (tRNA) A go-getter. Gets the right parts to make the right protein according to mRNA instructions amino acid attachment site Methionine U A C Anticodon UAC Ribosomal RNA (rRNA) • Part of the structure of a ribosome • Helps in protein production • Ribosomes contain two major rRNAs and 50 or more proteins • rRNA sequences are widely used for working out evolutionary relationships among organisms Type Size Large subunit (rRNAs) Small subunit (rRNA) prokaryotic 70S 50S (5S : 120 nt, 23S : 2906 nt) 30S (16S : 1542 nt) eukaryotic 80S 60S (5S : 121 nt, 5.8S : 156 nt, 28S : 5070 nt) 40S (18S : 1869 nt[4]) Ribosomes 40s 30s PRO 70S 30S 16S EUK 80S 50s 50S 5S 23 60s 40S 18S 60S 5.8S 5S 28S Ribosomes in Endoplasmic Reticulum Ribosomes Large subunit P Site A Site mRNA A Small subunit U G C U A C U U C G Initiation aa2 aa1 2-tRNA 1-tRNA anticodon U A C hydrogen bonds A U G codon G C U A C U U A C U G mRNA A Elongation peptide bond aa3 aa1 aa2 3-tRNA 1-tRNA anticodon U A C hydrogen bonds A U G codon G 2-tRNA G C A U U A C U U A C mRNA G A A Elongation peptide bond aa1 aa2 1-tRNA anticodon hydrogen bonds U A A codon 3-tRNA 2-tRNA C U aa3 G G A U G A A C U A C U U C mRNA G A End Product • The end products of protein synthesis is a primary structure of a protein. • A sequence of amino acid bonded together by peptide bonds. aa5 aa2 aa1 aa3 aa4 aa199 aa200 Overview of Protein Synthesis Post Translational Modification and Regulation • Recognition of Signal Peptide • Glycosylation-addition of sugars to proteins destined to be membrane or secreted – “O” linked- serine/threonine in golgi – “N” linked - asparagine in ER • Proteolysis cleavage: Truncation • Disulfide bonds bridge • Attachment or binding of groups(NAD,Zn,Mg,FAD) • Folding • Assembly of multiple subunits • R -group modifications (see next slide) Modification of Protein Precursors R -Group Modifications • Phosphorylation (via kinase on -OH group of serine/threonine/ tyrosine) • Methylation • Acetylation (palmitylation C16 via thioester with cysteine, myristication C14 at amino-terminal glycine) • Isoprenation (farnesyl,guanosyl groups) • Hydroxylation Maturation of Human Pre-Pro-insulin • Pre-pro-protein – a protein precursor that contains a signal peptide sequence; it is a nonpolar sequence at the head of the growing polypeptide chain and contains many hydrophobic amino acids residues. – required for its transfer into the cistern of the endoplasmic reticulum; the signal sequence is then cleaved to form the protein or proprotein. Insulin Protein Precursors: Pro-Insulin • Pre-pro-sequence – About 30 non-polar aa guide the protein to be secreted out of cells or into different compartment of the cell sub-organells • Pro-sequences – areas in the protein that are essential for its correct folding – usually in the transition of a protein from an inactive to an active state. – Pro-sequences may also be involved in pro-protein transport and secretion Post Translational Modification of Insulin Clinical Usage of C peptide Measurement • Patients with diabetes may have their C-peptide levels measured as a means of distinguishing type 1 diabetes from type 2 diabetes or Maturity onset diabetes of the young (MODY). • Measuring C-peptide can help to determine how much of their own natural insulin a person is producing as C-peptide is secreted in equimolar amounts to insulin. • C-peptide levels are measured instead of insulin levels because C-peptide can assess a person's own insulin secretion even if they receive insulin injections, • Because the liver does not metabolize C-peptide, meaning blood C-peptide may be a better measure of portal insulin secretion than insulin itself. • A very low C-peptide confirms Type 1 diabetes and insulin dependence and is associated with high glucose variability, hyperglycaemia and increased complications. Genetic Codon Change Causes Mutation in Proteins • Point Mutations – No change-silent due to alteration in 3rd base of codon, wobble or degenerate base – Missense- change in base leads to change in aa – Nonsense-formation or modification of termination codon • Frameshift- insertion or deletion of a nucleotide Diseases Related to Mutations • a-thalassemia (Nonsense) – normally 142 aa long – If stop codon at 142 mutates get a 172 aa version including these variants: • • • • Constant Spring: glutamine @ 142 Icaria: lysine @ 142 Seal Roe: glutamate @ 142 Koya Dora: serine @ 142 • Thalassemia- Frameshift Mutation – Abnormal Hemoglobin Wayne- everything after 138 is incorrect (goes to 147 before stop) Inhibitors of Protein Synthesis • Streptomycin / Gentamycin: 30S prok. initiation • Erythromycin: Target 50S prok. Elongation, both Gram + and Gram bacteria • Chloramphenicol: 70S ribosomal subunit in prok, elongation, broad spectrum, bone marrow suppression • Cyclohexamide: 80S euk translocation step, and fungus etc • Tetracycline: Inhibits incoming tRNA to A site at 30S subunit in prokaryotes • Puromycin: Premature terminator both prok and euk, mimic tRNA binds at A site, resistant to hydrolysis • Diphtheria toxin: Euk elongation factor II inhibitor • Note: Use of prokaryotic antibiotics can effect mitochondrial eukaryotic RNA processes; may be dangerous Antibiotics Bind to Ribosome • The following antibiotics bind to the 30S subunit of the ribosome: – Aminoglycosides – Tetracyclines • The following antibiotics bind to the 50S ribosomal subunit: – Chloramphenicol – Erythromycin – Streptogramins- a group of cyclic peptide antibiotics that inhibit, like macrolides and lincosamides, the synthesis of bacterial proteins.