CMB 33-34 Translation BL 2023 V2.pptx

Translation Bindong Liu, PhD Professor, Microbiology, Immunology and Physiology Meharry Medical College Email: [email protected]; Tel: 615-327-6877 September 2023 Lecture Objectives At the end of this lecture, students should be able to: • Describe the genetic code and facts about it • Describe the pro...

Translation Bindong Liu, PhD Professor, Microbiology, Immunology and Physiology Meharry Medical College Email: [email protected]; Tel: 615-327-6877 September 2023 Lecture Objectives At the end of this lecture, students should be able to: • Describe the genetic code and facts about it • Describe the process of translation and the components involved in the process • Describe the translation quality control and protein folding Flow of Genetic Information Molecular Biology of the Cell (© Garland Science 2015) The final product of gene expression is a polypeptide chain of amino acids whose sequence was prescribed by the genetic code. Genetic Code • The protein-coding information contained in DNA is a linear message • The information from DNA is copied and processed to an RNA form through transcription • The information is converted into a protein sequence through translation Figure 6-48 Molecular Biology of the Cell • A set of 3 nucleotides/bases corresponding to a single amino acid - codon • The sequence of bases (codons) determines the primary sequence of amino acids in a protein Genetic Code • There are 64 (4X4X4) possible triplet codes, but only 20 amino acids • As seen in the table, more than one triplet may code for the same amino acid • Note that several codons can also act as start (AUG) or stop (UAA) signals Genetic Code • Each codon specifies a single amino acid • More than one codon can specify the same amino acid Termed the degeneracy or redundancy of codons • Some aa correspond to a single codon AUG—initiator codon, methionine (Met, M) UGG–Tryptophan (Trp, W) • Often, codons encoding the same aa differ only at the 3rd nucleotide, also called the “wobble base” • Codon usage differs between nuclear and mitochondrial genes • Codon usage is consistent across species • Codons are read in non-overlapping groups called “reading frames” Main Players in Translation • mRNA transcribed from genomic DNA – already discussed • tRNA to transport amino acids • Ribosomes to “read” mRNA, align amino acids attached to tRNA and create the peptide bonds between adjacent amino acids Transfer RNA (tRNA) • The molecular link between the mRNA code and the sequence of amino acids is tRNA • ~ 80 nucleotides in length • L-shaped molecule • Contains unusual bases (Ψ, D) produced by chemical modification • Anticodon-complementary to codon on mRNA Molecular Biology of the Cell (© Garland Science 2015) • 3’ short single-stranded region (CCA) attaches to aa Transfer RNA (tRNA) (cont.) • Humans have nearly 500 tRNA genes, and among them, 48 different anticodons are represented • The anticodons of the tRNAs each have a complimentary codon in the mRNA. For example, the codon AUG would be the complement of the anticodon UAC Wobble Base-pairing • Only the wobble position permits wobble base-pairing • Only conventional base-pairing is allowed in positions 1 and 2 • The unconventional base pairings are generally weaker than conventional ones • Wobble base-pairing is different between bacteria and eukaryotes Molecular Biology of the Cell (© Garland Science 2015) • Due to wobble base-pairing, some tRNA molecules can base-pair with more than one codon e.g., tRNA with wobble position anticodon “I” will pair with U, C and A codons in bacteria and U and C in eukaryotes Activation of Amino Acid • Amino acids cannot be incorporated into protein unless they have been attached to the – CCA end of the correct tRNA • Aminoacyl-tRNA synthetases catalyze the attachment of aa to its appropriate tRNA • One synthetase for each amino acid Molecular Biology of the Cell (© Garland Science 2015) • tRNA synthetases are able to correct errors, i.e., they make sure that correct aa is attached to tRNA- therefore, have high specificity Ribosomes • Ribosomes are the ‘decoding’ units of the cell • Made of more than 50 different proteins and RNA molecules (rRNA) • Ribosomes consist of two major components The small subunit reads the RNA, The large subunit catalyzes the formation of the peptide bonds • Subunits are separate in the cytoplasm until they join to begin translation Molecular Biology of the Cell (© Garland Science 2008) • Ribosomes have binding sites for both tRNA and mRNA molecules A Comparison of Bacterial and Eukaryotic Ribosomes Molecular Biology of the Cell (© Garland Science 2015) Despite differences in the number and size of their rRNA and protein components, both bacter and eukaryotic ribosomes have nearly the same structure, and they function similarly. RNA-binding Sites in the Ribosome Exit • Three tRNA binding sites Peptidyl-tRNA Aminoacyl-tRNA E, P and A sites • One mRNA binding site • A tRNA molecule is held tightly at the A and P sites only if its anticodon forms base pairs with a complementary codon on the mRNA molecule Molecular Biology of the Cell (© Garland Science 2015) Translation There are three main steps in the process of Translation: • Initiation • Elongation • Termination Initiation • Common for both prokaryotes and eukaryotes obegins with a specific initiating tRNA obegins with the codon “AUG”- methionine • In eukaryotes oInitiator tRNA–methionine complex (Met–tRNAi) is loaded into the small ribosomal subunit along with additional proteins called eukaryotic initiation factors, or eIFs. oMet-tRNA is the only aminoacyl-tRNA that can tightly bind to small ribosomal subunit without the help of large subunit oMet-tRNA is also the only aa-tRNA that directly binds to the P site oNext, the small ribosomal subunit binds to the 5ʹ cap oThe small ribosomal subunit then moves forward (5ʹ to 3ʹ) along the mRNA scanning for AUG o90% starts translation at 1st AUG oSometime starts at 2nd or 3rd AUG oThe initiation factors dissociate and assemble with large subunit to start elongation Initiation (cont.) • In prokaryotes oNo 5’-cap omRNA contains a specific ribosome-binding site (called the Shine–Dalgarno sequence) a few bp upstream of AUG oA bacterial ribosome can assemble directly on a start codon that lies in the interior of an mRNA molecule, so long as a ribosome-binding site precedes it by several nucleotides. As a result, bacterial mRNAs are often polycistronic—that is, they encode several different proteins, each of which is translated from the same mRNA molecule Elongation Repeat step 1 • Ribosome translocates by three bases after peptide bond formed • New charged tRNA aligns in the A site • Peptide bond between amino acids in A and P sites is formed • Ribosome translocates by three more bases • The uncharged tRNA in the P site is moved to the E site and released • Elongation is in N→C direction Figure ©2010 PJ Russell, iGenetics 3rd ed • an amino group of incoming aa-tRNA (in the A site) carries out a nucleophilic attack on the esterified carboxyl group of peptidyl-tRNA (in the P site) catalyzed by peptidyltransferase Elongation Factors Drive Translation Forward and Improve Its Accuracy • Two elongation factors enter and leave the ribosome during each cycle, hydrolyzing GTP to GDP oBacteria: EF-Tu and EF-G oEukaryotes: EF1 and EF2 • EF-Tu recruits charged tRNA to A site (Requires hydrolysis of GTP) and increases the accuracy of translation • Accuracy in translation requires an expenditure of free energy • The binding of EF-G to the ribosome and the subsequent hydrolysis of GTP leads to a rearrangement of the ribosome structure, moving the mRNA being decoded exactly three nucleotides Molecular Biology of the Cell (© Garland Science 2008) Termination • Elongation proceeds until the STOP codon is reached UAA, UAG, UGA • No tRNA normally exists that can form base pairing with a STOP codon • Stop codons signal to the ribosome to stop translation • Recognized by a release factor • tRNA charged with the last amino acid will remain at the P site • Release factors cleave the amino acid from the tRNA • Ribosome subunits dissociate from each other Figure 6-72 Molecular Biology of the Cell (© Garland Science 2008) Polyribosome • Complex of an mRNA molecule and two or more ribosomes that translate mRNA instructions into polypeptides • How? - As soon as the preceding ribosome has translated enough of the nucleotide sequence to move out of the way, the 5ʹ end of the mRNA is threaded into a new ribosome • Ribosomes can be spaced as close as 80 nucleotides apart along a single mRNA molecule • Both bacteria and eukaryotes use polysomes to speed up protein synthesis Figure 6-73 Molecular Biology of the Cell (© Garland Science 2008) Inhibitors of Prokaryotic Protein Synthesis Are Useful as Antibiotics • Many of the most effective antibiotics used in modern medicine are compounds made by fungi that inhibit bacterial protein synthesis • Some of these drugs interfere preferentially with the function of bacterial ribosomes. Low toxicity to humans. • Most of the antibiotics shown bind directly to pockets formed by the ribosomal RNA molecules. o Hygromycin B induces errors in translation o Spectinomycin blocks the translocation of the peptidyl-tRNA from the A site to the P site o Streptogramin B prevents the elongation of nascent peptides. Quality Control • Quality control mechanisms act to prevent translation of damaged mRNAs • Errors possible o Incorrectly or incompletely processed mRNAs are inadvertently sent to the cytosol o Correct mRNAs can be broken or otherwise damaged in the cytosol • Mechanism for quality control o Avoid translating broken mRNAs o Nonsense-mediated mRNA decay Nonsense-mediated mRNA Decay • Eliminates defective mRNAs before they move away from the nucleus • Determines that an mRNA molecule has a nonsense (stop) codon (UAA, UAG, or UGA) in the “wrong” place • Ribosome translates mRNA as its 5’ end emerges from a nuclear pore • Moving ribosomes remove exon junction complexes (EJCs) • If no EJCs remain on mRNA when translation stops, the mRNA passes QC • If EJCs remain on mRNA when translation stops, the mRNA fails QC and will be rapidly degraded Protein Folding • Newly translated polypeptide is not ready for function • To be ready – the polypeptide chain must fold up into its unique 3D conformation, assemble correctly with the other protein subunits (if required), bind other cofactors or be modified by protein kinases or other enzymes as required for its activity, • Molecular chaperones are proteins that guide the folding of the nascent polypeptide into its final functional form • Molecular chaperones specifically recognize incorrect, off-pathway configurations by their exposure to hydrophobic surfaces, which incorrectly folded proteins are typically buried in the interior Co-translational Protein Folding Figure 6-79 Molecular Biology of the Cell (© Garland Science 2008) Molecular Chaperones Help Guide the Folding of Most Proteins Figure 6-81A Molecular Biology of the Cell (© Garland Science 2008) • Most proteins probably do not fold correctly during their synthesis and require a special class of proteins called molecular chaperones to do so • Correctly folded proteins are typically buried on hydrophobic surfaces in the interior. • Molecular chaperones specifically recognize and correct the incorrect, offpathway configurations by their exposure to hydrophobic surfaces The Processes That Monitor Protein Quality Following Protein Synthesis. • Some protein fold correctly and assembles on its own with its partner proteins • Incompletely folded proteins are helped to properly fold by molecular chaperones: o First by a family of hsp70 proteins o Some cases by hsp60 like proteins Figure 6-82 Molecular Biology of the Cell (© Garland Science 2008) • Proteolytic machinery marks protein for degradation by proteasome • Proteolytic machinery and the chaperones compete with one another to recognize a misfolded protein • The combined activity of all of Protein Production in a Eukaryotic Cell Figure 6-87 Molecular Biology of the Cell (© Garland Science 2008) The production of a protein by a eukaryotic cell Many levels of regulation/variation True or false 1. The large and small ribosome subunits are normally separated in the cytoplasm until they are used for translation 2. Met-tRNA binds to both large and small ribosomal subunits to initiate the translation 3. Met-tRNA ribosomal complex only binds to mRNA with intact 5’-cap and 3’ poly-A to initiate the translation 4. Nonsense-mediated mRNA decay is a process of preventing the translation of mRNA with no 5’-cap 5. Release factor facilitates termination of the translation 6. Each type of tRNA molecule can only attach to one type of amino acid 7. 5´-methylguanosine cap addition is the first modification to occur in eukaryotes 8. Introns are spliced together while exons are removed during mRNA processing