CH 15 Translation

Genetic Code and Translation Lecture 14 Outline Ribosomes and Transfer RNAs (tRNAs) Three Steps of Translation Genetic Code Variation in Protein Structure Provides the Basis of Biological Diversity Posttranslational Modification Proteins Function in Many Diverse Roles Functional Domains Ribosomes Are Translation Machines Polypeptides are strings of amino acids that are assembled by ribosomes Ribosomes are “machines” that contain multiple ribosomal RNAs (rRNAs) and proteins Ribosomes translate mRNA in the 5?–3? direction, reading each triplet codon and assembling the amino acids in the order specified by the codons Bacterial and Eukaryotic Ribosome Structures Ribosomes in bacteria and eukaryotes perform three tasks Bind mRNA and identify the start codon, where translation begins Facilitate complementary base pairing of mRNA codons and the corresponding tRNA anticodons Catalyze formation of peptide bonds between amino acids on the growing polypeptide chain Ribosome Composition Ribosome composition—number and sequence of rRNA molecules and number and type of proteins—differs between bacteria 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 Important Regions of Ribosomes 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 Ribosomes also form a channel from which the polypeptide chain emerges 3D Structure of the Ribosome Ribosomes are 25 nm in diameter and powerful imaging techniques are needed to explore their configurations Cryo-electron microscopy uses liquid nitrogen or liquid ethane to freeze macromolecules, preserving their native state A frozen macromolecule is then placed on a micro-caliper and scanned from various angles using an electron beam to create a three-dimensional picture Translation Occurs in Three Phases Initiation Elongation Termination The phases are similar in bacteria and eukaryotes, though there are several differences Initiation Initiation begins when the small ribosomal subunit binds near the 5? end of the mRNA and identifies the start codon The initiator tRNA, carrying the first amino acid of the polypeptide, binds to the start codon Finally, the large subunit joins the small subunit to form the intact ribosome and translation begins Initiation factor proteins help control ribosome formation and binding of the initiator tRNA GTP provides the energy for initiation tRNAs used during translation are called charged tRNAs, whereas tRNAs without amino acids attached are uncharged Prokaryotic Translational Initiation In E. coli, six molecular components come together to initiate translation: mRNA, the small ribosomal subunit, the large subunit, the initiator tRNA, three initiation factor proteins, and GTP For most of initiation, the 30S subunit is affiliated with an initiation factor, IF3, which prevents the 30S subunit from binding the 50S subunit The small subunit-IF3 complex binds near the 5? end of the mRNA, searching for the start codon The Shine-Dalgarno Sequence The preinitiation complex forms when the 16S rRNA and the Shine-Dalgarno sequence on the mRNA base pair The Shine-Dalgarno sequence is a purine-rich sequence of about six nucleotides three to nine nucleotides upstream of the start codon A complementary pyrimidine-rich sequence is found near the 3? end of the 16S rRNA Examples of Shine Dalgarno Prokaryotic Initiation: Step Two 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, N-formylmethionine (fMet); the charged initiator tRNA is called tRNAfMet Initiation factor, IF-2, and a GTP molecule are bound to the tRNAfMet and IF-1 joins the complex; together these form the 30S initiation complex Prokaryotic Initiation: Final Step 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 Eukaryotic Translational Initiation The eukaryotic 40S ribosomal subunit complexes with eukaryotic initiation factor (eIF) proteins eIF1A, eIF3, and a charged tRNAmet bind the small subunit to form the preinitiation complex This complex is recruited to the 5? cap region of mRNA The preinitiation complex joins a group of at least four eIF4 proteins that assembles at the 5? cap of the mRNA Together all of these components comprise the initiation complex Once the initiation complex is formed, it uses scanning to move the small subunit along the 5? UTR in search of the start codon ATP hydrolysis is required for this process Eukaryotic Initiation: Final Steps The correct start codon (AUG) can be located because it is embedded in a consensus Kozak sequence: 5?-ACCAUGG-3? Location of the start codon leads to recruitment of the 60S subunit to the complex, using energy from GTP hydrolysis, and the dissociation of the eIF proteins Elongation Elongation begins with recruitment of elongation factor (EF) proteins that use energy of GTP hydrolysis to Recruit charged tRNAs to the A-site Form peptide bonds between sequential amino acids Translocate the ribosome in the 3? direction along the mRNA tRNA Molecules and Charged tRNAs tRNA molecules are transcribed from tRNA genes Correct charging of each tRNA molecule is crucial for the integrity of the genetic code Enzymes called aminoacyl-tRNA synthetases or tRNA synthetases catalyze the addition of the correct amino acid to tRNAs Recognition of the iso-accepting tRNAs by the enzyme is complex with no single set of rules tRNA Synthetase tRNA synthetase is a large molecule that contacts several points on the tRNA in the recognition process The acceptor stem of the correct tRNA fits into the active site of the enzyme The active site contains the amino acid to be added to the tRNA; ATP provides the energy for amino acid attachment tRNA synthetase uses a proofreading system to maintain a very low error rate Prokaryotic Elongation Several different EFs and other ribosomal proteins carry out elongation in a series of steps Charged tRNAs affiliated with an EF inspect the open A-site; the tRNA with the correct anticodon sequence enters the A-site The enzyme peptidyl transferase then catalyzes peptide bonds between amino acids at the P- and A-sites Additional Steps of Elongation The elongated polypeptide is transferred to the tRNA at the A-site while the tRNA from the P-site then exits through the E-site EFs then translocate the ribosome, moving it three nucleotides toward the 3? end of the mRNA This moves the tRNA at the A-site to the P-site and opens a new A-site at the next codon Elongation: Eukaryotic Distinct elongation factors carry out elongation in eukaryotes, and the steps are similar to those of bacteria Many different charged tRNAs inspect the A-site Only the one with the anticodon sequence that is complementary to the codon sequence can bind the A-site Eukaryotic Elongation Is Very Similar to Bacterial Elongation Peptidyl transferase catalyzes peptide bond formation This transfers the polypeptide chain to the tRNA on the A-site from the tRNA on the P-site Translocation moves the ribosome along to the next codon on the mRNA so that the new A-site is ready to receive the next tRNA Termination The elongation cycle continues until one of the three stop codons (UAA, UAG, UGA) enters the A-site of the ribosome Bacteria and eukaryotes both 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 when the GTP complexed to the RF is hydrolyzed Termination: Release Factors In bacteria, the release factor RF1 recognizes UAG and UAA and RF2 recognizes UAA and UGA Eukaryotic termination is accomplished by a single release factor called eRF, which recognizes all three stop codons 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 The Triplet Code Groups of three consecutive nucleotides (codons) in an mRNA each correspond to one amino acid The genetic code contains 64 different codons; with only 20 common amino acids, this leads to redundancy–some amino acids are specified by more than one codon The Genetic Code Displays Third-Base Wobble The triplet genetic code, with 64 combinations, provides enough variety to code 20 amino acids 61 codons specify amino acids, and 3 are stop codons All amino acids except methionine and tryptophan are specified by at least two codons, called synonymous codons Third-Base Wobble Though there are 61 different codons that specify amino acids, most genomes have 30-50 different tRNA genes A relaxation of the strict complementary base-pairing rules at the third base of the codon is called third-base wobble tRNA molecules with different anticodons for the same amino acids are called iso-accepting tRNAs Most synonymous codons can be grouped into pairs that differ only in the third base; the pairs either both carry a purine (A or G) or both carry a pyrimidine (C or U) Third-base wobble occurs through flexible pairing at the 3?-most nucleotide of the codon and the 5?-most nucleotide of the anticodon However, a pyrimidine must still base-pair with a purine Experiments Deciphered the Genetic Code A remarkable set of experiments in the 1960s deciphered the genetic code and answered the following questions: Do neighboring codons overlap one another? How many nucleotides make up an mRNA codon? Is the polypeptide-coding information of mRNA continuous or does it contain gaps? No Overlap in the Genetic Code If the genetic code were nonoverlapping, each mRNA nucleotide would be part of a single codon If the code were overlapping, each nucleotide could be part of multiple codons In 1957, Sidney Brenner determined that an overlapping code was not possible because it was too restrictive – the sequence of one codon would limit the possible sequences for the two subsequent codons In 1960, a study of single nucleotide substitutions by Fraenkel-Conrat and colleagues showed that single nucleotide changes led to single amino acid changes An overlapping code would have led to multiple amino acid changes as a result of altering one nucleotide Extended the Analysis 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) A Triplet Genetic Code Proof of a triplet genetic code came in 1961 when researchers (Crick, Barnett, Brenner, and Watts-Tobin) created mutations by insertion or deletion of single nucleotides This leads to a change in reading frame of the mRNA Reading frame refers to the specific codon sequence as determined by the start codon Frameshift Mutations Mutations that alter reading frame are called frameshift mutations and garble the sense of the translated message Wild type: YOU/MAY/NOW/SIP/THE/TEA Mutant addition: YOU/MAC/YNO/WSI/PTH/ETE/A Mutant deletion: YOU/MAY/NOS/IPT/HET/EA All the codons after the addition or deletion will specify the wrong amino acids Reversion of Frameshift Mutations Frameshift mutations may be restored if a second mutation in a different location in the gene restores part of the reading frame Mutant addition: YOU/MAC/YNO/WSI/PTH/ETE/A Reversion mutant deletion: YOU/MAC/YNO/SIP/THE/TEA The frameshift is now confined to just a small area between the original mutation and the reversion mutation – the rest of the protein (sentence) is normal No Gaps in the Genetic Code Crick and colleagues suggested that the genetic code is read continuously, with no gaps, spaces, or pauses between codons For example, if a spacer were present a transcript might read: YOUxMAYxNOWxSIPxTHExTEAx; if the spacer existed, an inserted or deleted nucleotide would affect only one codon YOUxMATYxNOWxSIPxTHExTEA First Steps in Deciphering the Genetic Code Nirenberg and Matthai performed an experiment in 1961 that laid the groundwork for later work Strings of repeating nucleotides were translated in vitro and the resulting polypeptide identified For example, an artificial mRNA containing only uracils, poly(U), resulted in polypeptides containing only phenylalanine, so the codon UUU corresponds to the amino acid Phe The Final Piece of the Puzzle Nirenberg and Leder used mini-RNAs just three nucleotides long (one for each possible codon) to resolve the ambiguities of previous experiments They added the mini RNAs to in vitro translation systems–each system contained all amino acids with one of these labeled with 14C They isolated the ribosome-tRNA-mRNA complexes and determined which mini-RNA was associated with each labeled amino acid 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 Translation and Transcription In bacteria, the coupling of transcription and translation allows ribosomes to begin translating mRNAs that have not yet been completed In eukaryotes, mRNAs are produced in the nucleus They must be processed to form mature mRNAs and then exported to the cytoplasm for translation Each polypeptide-producing gene in eukaryotes produces monocistronic mRNA, an RNA that directs synthesis of a single kind of polypeptide Groups of bacterial genes, called operons, 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 Polycistronic mRNA Polycistronic mRNAs consist of multiple polypeptide- producing segments, each of which contains a Shine-Dalgarno site for ribosome binding and initiation of translation An intercistronic spacer sequence that is not translated separates the segments When the spacer sequences are short (? 6 nucleotides), ribosomes may proceed to the next start codon after finishing translation of the previous segment Ribosomes and Transfer RNAs (tRNAs) (Subunits and sites in the ribosome) Three Steps of Translation (i.e. initiation) Genetic Code (i.e. triplet, reading frame etc) Posttranslational Modification, Proteins Function in Many Diverse Roles, Functional Domains Operons and Polycistronic RNA (help for understanding gene regulation after spring break) Take Home Message