Lecture 22 - Gene Expression Part 2 PDF

2  Overall goal: Convert the sequence of bases in the ________ RNA to a sequence of ________ Amino acids in a polypeptide. Major Players: A. ________...

2  Overall goal: Convert the sequence of bases in the ________ RNA to a sequence of ________ Amino acids in a polypeptide. Major Players: A. ________ mRNA acts as the message. B. ________ Amino acids are the building blocks. C. ________ tRNA act as adaptor molecules. Direction of Translation D. ________ Ribosome act as the biological catalyst. 3 Direction of Translation 4  The genetic code contains the rules that specify the relationship between a sequence of nucleotide bases in _______ mRNA and the corresponding sequence of amino acids in a _______ Polypeptide. A set of _______ 3 nucleotides encodes a specific amino acid or stop signal. This triplet sequence is referred to as a _______ Codon. 5 1. It is ___________ Redundant. All amino acids except two are encoded by more than one codon. 2. It is ___________ Specific. One codon never codes for more than one amino acid. 3. It is ___________ Non-ambiguous. The first two bases are usually identical when multiple codons specify the same amino acid. 4. It is nearly ___________ Universal. With a few minor exceptions, all codons specify the same amino acids in all organisms. 6 What are these adaptor molecules? 7  Speaks both DNA language and protein language. Acts as an adapter molecule and “decodes” the code. Links a codon to a specific amino acid using its anticodon. 8  3! Amino acid attachment site 5! Small RNA molecules (approx. 80 Acceptor stem nts) that are post-transcriptionally modified. Forms a cloverleaf: 3 hairpin (stem- loop) structures through intramolecular base-pairing. * Modified Hydrogen nucleotides bonds The 5’-CCA-3’ sequence at the 3' end of each tRNA is the attachment site for amino acids. The triplet on the loop at the Anticodon opposite end is the anticodon that base pairs with the mRNA codon. (a) Two-dimensional structure 9  All tRNAs have a similar tertiary structure. The secondary structure of tRNA folds over to produce an upside-down L- shaped tertiary structure. 10  A tRNA that is charged is covalently linked to its corresponding amino acid and is called an aminoacyl tRNA. Enzymes that charge the tRNA are called aminoacyl tRNA synthetases. ATP is required to attach an amino acid to RNA. For each of the 20 amino acids, there is a different aminoacyl tRNA synthetase that can add that amino acid to one or more tRNAs with the correct anticodon. 11  Active Site Each enzyme is specific for one amino acid. Recognizes the correct tRNA by interacting with the anticodon. 12  tRNA Aminoacyl-tRNA Steps: Charging synthetase (enzyme) 1. Amino acid + ATP è Aminoacyl-AMP + pyrophosphate 2. Aminoacyl-AMP + tRNA è Aminoacyl-tRNA + AMP Amino acid P Adenosine P P P Adenosine P Pi ATP Aminoacyl-tRNA Pi Pi tRNA synthetase tRNA Amino acid P Adenosine Computer model What type of works is being Aminoacyl tRNA carried out here? Explain how (“charged tRNA”) energy coupling applies here. 13 1. Binds mRNA. 2. Provides a binding site for tRNA. 3. Catalyzes the formation of peptide bonds. 14  A ribosome is a complex of _________ Proteins and ribosomal _________ RNA (rRNA). Ribosomes can be separated into two major subunits: A. Small subunit B. Large subunit 15  Bacterial and eukaryotic ribosomes are structurally and functionally similar but have significant differences. Some antibiotic drugs specifically target bacterial ribosomes without harming eukaryotic ribosomes. 16  The ribosome contains three tRNA binding sites. A. The A (aminoacyl) site. B. The P (peptidyl) site. C. The E (exit) site. 17 18  Translation has three phases: A. Initiation B. Elongation C. Termination All three stages require proteins called translation factors that aid in the process. All three stages require energy in the form of GTP. The following is the translation mechanism in bacteria. 19  Initiation is a three-step process: Ribosome binding site Start mRNA codon Initiation factors Small subunit of ribosome I1. mRNA binds to small subunit of ribosome 20  Aminoacyl tRNA I2. f-Met tRNA binds 21 Large subunit of ribosome I3. Large subunit binds 22  Elongation is a three-step process: Ribosome Peptidyl site tRNA 2. 1. Exit site Aminoacyl site mRNA E1. Incoming aminoacyl tRNA 23  Peptide bond Ribosome’s active site 1. 2. E2. Peptide bond formation 24  1. 2. E3. Translocation 25  3. 1. 2. E1. Incoming aminoacyl tRNA 26  1. 2. 3. E2. Peptide bond formation 27 Exit tunnel Elongation cycle continues 1. Next tRNA anticodon? 2. 3. E3. Translocation (and Ejection) 28  The termination phase starts when the A site encounters a stop codon. This causes a protein called a release factor to enter the site. The release factor resembles tRNAs in size and shape but do not carry an amino acid. This factor catalyzes the hydrolysis of the bond linking the tRNA in the P site with the polypeptide chain and releases the polypeptide. The complex dissociates. 29  Termination is a three- step process: Hydrolysis of bond linking tRNA and polypeptide tRNA Release factor mRNA STOP codon T1. Release factor binds to stop codon 30 mRNA T2. Polypeptide is released by hydrolysis 31  Large subunit Release factor mRNA Small subunit T3. Dissociation of complex 32  Translation of polypeptides alone is not sufficient to make functional proteins. Several steps are (may be) required to process a polypeptide into a functional protein: 1. Folding via molecular chaperones 2. Binding with other subunits to form a protein 3. Posttranslational modifications examples? 4. Association with cofactors 33 Genome < Transcriptome < Proteome 34  To carry out its function, a protein must be transported to the appropriate cellular location or outside of the cell. This is called protein targeting and requires a targeting signal. Targeting signal is an amino acid sequence (signal peptide) or a chemical modification to the folded protein. The signal is recognized by a specific targeting system and the protein is transported to a specific location. 35 1. Cytosol 3 2. Organelles 2B A. Nucleus B. Endomembrane 2A system C. Semiautonomous organelles 1 3. Plasma membrane 2C 4. Outside of the cell 4 36 37