Chapter 9. Genetic Code and Translation

Chapter 9. Genetic Code and Translation The Genetic Code: How many bases of mRNA code for a single amino acid? There are 20 amino acids in cellular proteins If: 1 base = 4 aa  not enough codes 2 bases = 4x4=16 aa  not enough codes 3 bases = 4x4x4=64 aa more than enough -- therefore, at least thre...

Chapter 9. Genetic Code and Translation The Genetic Code: How many bases of mRNA code for a single amino acid? There are 20 amino acids in cellular proteins If: 1 base = 4 aa  not enough codes 2 bases = 4x4=16 aa  not enough codes 3 bases = 4x4x4=64 aa more than enough -- therefore, at least three nucleotides. Is the code overlapping or non-overlapping? (mRNA) A Triplet Genetic Code  Proof of a triplet genetic code came in 1961 when researchers (Francis Crick, Leslie Barnett, Sydney Brenner, and R.J. Watts-Tobin) created mutations by insertion or deletion of single base pairs in the rII gene in T4 bacteriophage  This leads to a change in the reading frame of the mRNA  Reading frame refers to the specific codon sequence as determined by the start codon 4 Frameshift Mutations 1. rII locus of phage T4 2. Non-mutants can grow in and lyse E. coli K12 and E. coli B 3. Mutants of this gene cannot lyse E. coli strain K12 but can infect and lyse E.coli B This sets up an elegant test system for mutational analysis E coli K12 rII+ E coli B Plaques in an E coli lawn Wild type E coli K12 rII+ E coli B lyse E. coli K12 and E. coli B E coli K12 Mutant Tests presence of mutant rIIE coli B lyse E. coli B Can be used for mutational studies Generate frameshift mutations by proflavin E coli K12 rII- rIIE coli B Deletion mutant rII- Additional deletion mutation rII- rII- Frameshift mutations produced by proflavin Crick et al., conclusively showed that it took three insertions or deletions to restore a reading frame  genetic code consists of three successive nucleotides (triplet) The genetic code is read continuously, with no gaps, spaces, or pauses between codons Decipher the Genetic Codes First Steps in Deciphering the Genetic Code Marshall Nirenberg and Heinrich Matthaei, 1961 In vitro protein-synthesizing system:  an enzyme, polynucleotide phosphorylase, allowed the production of synthetic mRNAs (without DNA template)  ribosomes, tRNA, amino acids UUUUUUUUUU = polyphenylalanine It took five years to complete the code chart using mixed copolymers and repeating copolymers The four ribonucleotides in RNA RNA homopolymers – RNA nucleotides with only one type of ribonucleotide – RNA homopolymers were added to in vitro translation system – Helped decipher which amino acids were encoded by first few codons based on which amino acids were incorporated into polypeptide Polynucleotide phosphorylase – Enzyme that catalyzes production of synthetic mRNAs – mRNAs serve as template for in vitro (cell-free in test tube) system Use of synthetic mRNAs to determine genetic code possibilities Figure 9.18 Khorana Extended the Analysis of the Genetic Code  Har Gobind Khorana synthesized mRNA molecules with repeating di-, tri-, and tetranucleotides, and translated them in vitro to define many more codons  For example, a dinucleotide repeat (UC)n produces an mRNA with the sequence 5-UCUCUCUCUCUCUCUC-3 and two possible codons, UCU and CUC  The resulting polypeptides contained alternating amino acids, serine (Ser) and leucine (Leu) Example Polypeptide Production from Synthetic mRNAs Nirenberg and Leder’s Results  Nirenberg and Leder tested all 64 possible codons  They identified all 61 of the codon–amino acid associations  They also identified the three stop codons, UAA, UAG, and UGA The (Almost) Universal Genetic Code  In all organisms from bacteria to humans, the processes of transcription and translation are similar  Because the genetic code is universal, bacteria can be used to produce important proteins from plants and animals  However, there are a few exceptions to the universality of the genetic code, found principally in mitochondria, though there are two exceptions in living organisms Genomes Using Modifications of the Universal Genetic Code The Genetic Code synonymous codons The Genetic Code The code is degenerate. Of 20 amino acids, 18 are encoded by more than one codon. Met (AUG) and Trp (UGG) are the exceptions; all other amino acids correspond to a set of two or more codons called synonymous codons. Codon sets often show a pattern in their sequences; variation at the third position is most common. AUG is the usual start signal. Stop signals are codons with no corresponding tRNA. There are three stop codons: UAG, UAA and UGA. Deciphering the Genetic Code  The genetic code was deciphered between 1961 and 1965  It was a milestone in establishing the central dogma of biology: DNA RNA protein  Har Gobind Khorana and Marshall Nirenberg were awarded the Nobel Prize in physiology or medicine in 1968 for their contributions Alignment of DNA, mRNA, and polypeptide Figure 9.3 Translation Translation Animation Initiation of translation in prokaryotes -https://www.youtube.com/watch?v=KZBljAM6B1s Initiation of translation in eukaryotes – https://www.youtube.com/watch?v=qIwrhUrvX-k Translation overview  Translation – Biological polymerization of amino acids into polypeptide chains  Translation requires – amino acids – messenger RNA (mRNA) – transfer RNA (tRNA) – ribosomes Figure 9.2 Messenger RNA  The mRNA sequence dictates the resulting amino acid sequence  Boundaries of translation are defined by a start codon that corresponds to the N-terminus of the protein and a stop codon that corresponds to the C-terminus  The 5 untranslated region (5 UTR) and 3 UTR are segments of the mRNA outside of the translated regions tRNA – Adaptor Molecule  tRNAs: Transfer RNAs – Transcribed from DNA – Small in size and very stable – 75–90 nucleotides – tRNA anticodons complement mRNAs – tRNAs carry corresponding amino acids – Corresponding amino acid is covalently linked to CCA sequence at 3’ end of all tRNAs Cloverleaf structure of tRNA Corresponding amino acid is covalently linked to CCA sequence at 3 end of all tRNAs Charging tRNA  Aminoacylation: tRNA charging – Before translation can proceed, tRNA molecules must be chemically linked to respective amino acids – Aminoacyl tRNA synthetase  Enzyme that catalyzes aminoacylation  20 different synthetases, one for each amino acid  Highly specific; recognize only one amino acid Charging tRNA Figure 9.16 An aminoacyl-tRNA synthetase attaches an amino acid to its tRNA Third-Base Wobble  tRNA molecules with different anticodons for the same amino acids are called isoaccepting tRNAs  Though there are 61 different codons that specify amino acids, most genomes have 30 to 50 different tRNA genes  A relaxation of the strict complementary base-pairing rules at the third base of the codon is called thirdbase wobble Codon–anticodon pairing Third base wobble Figure 9.14 Figure 9.15 Ribosome Structures  Ribosomes in bacteria, archaea, and eukaryotes perform three tasks: 1. Bind mRNA and identify the start codon, where translation begins 2. Facilitate complementary base pairing of mRNA codons and the corresponding tRNA anticodons 3. Catalyze formation of peptide bonds between amino acids on the growing polypeptide chain Ribosomes – Consist of ribosomal proteins and ribosomal RNAs (rRNAs) – Consists of large and small subunits  Prokaryote ribosomes are 70S  Eukaryote ribosomes are 80S rRNA folds up by intramolecular base pairing (16s rRNA) Ribosome Composition  Number and sequence of rRNA molecules and number and type of proteins—differs between bacteria, archaea, and eukaryotes  Ribosomes are composed of two subunits, the large ribosomal subunit and the small ribosomal subunit  Ribosomal subunit size is measured in Svedberg units (S), a property based on size, shape, and hydration state Ribosomes of E. coli  The small subunit is 30S and contains 21 proteins and one 16S rRNA molecule  The large subunit is 50S and contains 32 proteins, a small 5S rRNA, and a large 23S rRNA  The fully assembled ribosome is 70S Eukaryotic Ribosomes  Mammalian ribosomes have the small (40S) subunit contains about 35 proteins and one 18S rRNA  The large subunit (60S) contains 45 to 50 proteins and three rRNA molecules, of 5S, 5.8S, and 28S  These ribosomes (80S) contain the P and E sites and the polypeptide channel Ribosomes of bacteria and eukaryotes Figure 9.4  The peptidyl site (P site) holds the tRNA to which the polypeptide is attached  The aminoacyl site (A site) binds a new tRNA molecule containing an amino acid to be added to the growing polypeptide chain  The exit site (E site) provides an avenue for exit of the tRNA after its amino acid has been added to the chain Three-dimensional computer interpretations of cryo-EM–generated data depict ribosome structure Figure 9.5 Translation of mRNA in prokaryotes 1. Initiation 2. Elongation 3. Termination Key sites of interaction in the ribosome A site: for aminoacyl-tRNA P site: for peptide-tRNA E site: for exit of tRNA Translation Initiation in Prokaryotes Figure 9.6  Shine-Dalgarno sequence (AGGAGG) – Precedes AUG start codon in bacteria – Base-pairs with region on 16S rRNA of 30S small subunit, facilitating initiation  Initiation complex – Small ribosomal subunit  initiation factors  mRNA at codon AUG – Combines with large ribosomal subunit The Shine–Dalgarno consensus binding sequence Figure 9.7 The Second Step of Initiation  The initiator tRNA binds to the start codon where the P site will be once the ribosome is fully assembled  The amino acid on the initiator tRNA is a modified amino acid, Nformylmethionine (fMet); the charged initiator tRNA is called tRNAfMet  Initiation factor, IF2, and a GTP molecule are bound at the P site to facilitate binding of tRNAfMet and IF1 joins the complex; together these form the 30S initiation complex The Final Step of Initiation  In the last stage of initiation, the 50S subunit joins the 30S subunit to form the intact ribosome  The union of the two subunits is driven by hydrolysis of GTP to GDP  The dissociation of IF1, IF2, and IF3 accompanies the joining of the subunits to create the 70S initiation complex Bacterial Translation Elongation Figure 9.9 Bacterial Translation Elongation Figure 9.9 Translation Termination  The elongation cycle continues until one of the three stop codons (UAA, UAG, UGA) enters the A site of the ribosome  All organisms use release factors (RF) to bind a stop codon in the A site  The polypeptide bound to the tRNA at the P site is then released while the RF is ejected and the ribosomal subunits separate Termination of translation by release factor (eRF) proteins. Figure 9.10 The Translational Complex  Cell biologists estimate that each bacterial cell contains about 20,000 ribosomes, collectively accounting for 25% of the mass of the cell  Electron micrographs reveal structures called polyribosomes, containing groups of ribosomes all actively translating the same mRNA  In bacteria, the coupling of transcription and translation allows ribosomes to begin translating mRNAs that have not yet been completed Polyribosomes Figure 9.11 Translation of Bacterial Polycistronic mRNA  Each polypeptide-producing gene in eukaryotes produces monocistronic mRNA, an RNA that directs synthesis of a single kind of polypeptide  Groups of bacterial and archaeal genes, called operons, often share a single promoter and produce polycistronic mRNAs that lead to synthesis of several different proteins  The genes of operons function in the same metabolic pathway and are regulated as a unit Eukaryotic Translation is more complex  Translation in eukaryotes – Requires more factors for initiation, elongation, and termination than in bacteria  Ribosomes are not free-floating; instead are associated with endoplasmic reticulum  Translation in eukaryotes – Ribosomes are larger and longer lived than bacteria – Transcription occurs in nucleus  5 end of mRNA capped with 7-methylguanosine residue at maturation, which is essential for translation  Poly-A tail added at 3 end of mRNA – Translation occurs in the cytoplasm Protein Structure  Four levels of protein structure – Primary: Sequence of amino acids – Secondary: -helix and -pleated sheets – Tertiary: Three-dimensional conformation – Quaternary: Composed of more than one polypeptide chains Peptide Bond – Dehydration (condensation) reaction facilitates bond between carboxyl group of one amino acid and amino group of another Secondary Structure © 2015 Pearson Education, Inc. Figure 14-18 Tertiary Figure 14-19 Quaternary Figure 14-20 Polypeptide Structure Protein Sorting  Polypeptides destined for secretion are produced at the rough ER  These polypeptides are transported into the cisternal space of the rough ER  Once inside, polypeptides are processed and packaged for transport to the Golgi apparatus  In the Golgi apparatus, proteins are further processed and then packed into vesicles for transport to their intercellular destination Protein Sorting Proteins enter the endoplasmic reticulum (ER). Antibiotics targeting protein synthesis Kanamycin, Streptomycin Gentamicin Nat Rev Microbiol. 2010 Jun; 8(6): 423–435. End