RNA Transcription & Translation PDF

RNA Transcription & Translation Cellular & Molecular Biology MD105 Dr C. Michaeloudes  RNA Transcription & Translation Dr C. Michaeloudes Cellular & Molecular Biology MD105  Lecture Objectives To understand: The basic mechanism of gene transcription...

RNA Transcription & Translation Cellular & Molecular Biology MD105 Dr C. Michaeloudes  RNA Transcription & Translation Dr C. Michaeloudes Cellular & Molecular Biology MD105  Lecture Objectives To understand: The basic mechanism of gene transcription The basic characteristics of the gene code The structure of ribosomes and their role in translation The basic mechanism of translation  From DNA to protein DNA DNA is transcribed into mRNA in the DNA replication nucleus Transcription mRNA is released from the nucleus into mRNA mRNA the cytoplasm through nuclear pores mRNA is attached to ribosomes, which mRNA translate it into a polypeptide chain Ribosome Polypeptide chains are folded into the Translation protein active conformation in the Polypeptide cytoplasm, mitochondria, nucleus and ER Protein folding Active protein Gene Transcription  Gene transcription The process by which the information in a DNA strand is copied into an RNA molecule This process is catalyzed by RNA Polymerase enzymes  Gene transcription RNA 5’ 3’ U G G U A G A C C A T C A G 3’ 5’ Template DNA RNA Polymerase RNA polymerase adds nucleotides to the growing RNA chain and catalyzes the formation of phosphodiester bonds between them  RNA Polymerases Large multi-subunit enzymes In eukaryotes there are three RNA Polymerases: – RNA Pol I - transcribes ribosomal RNA (rRNA) – RNA Pol II - transcribes mRNA and most non-coding RNAs – RNA Pol III - transcribes tRNA & other Prokaryotes use only one RNA Polymerase  RNA Polymerase II Transcribes mRNA Large multiprotein complex consisting of 12 subunits (RBP1-12) RPB1 - DNA-directed RNA polymerase II subunit catalyzes transcription of DNA to mRNA Ø Phosphorylation of RPB1 regulates enzyme activity Cannot “read” DNA – requires transcription factors for DNA binding and transcription Can start creating an RNA chain without a primer RNA Polymerase II transcription  1. Transcription Initiation Transcription TF TF GENE RNA Pol II P RNA Pol II binds to a non-coding DNA sequence immediately before the target gene called the core promoter RNA Pol II cannot “read” the DNA sequence to find where to bind Guided to the core promoter by proteins called transcription factors (TF) RNA Pol II and transcription factors form the pre-initiation complex RNA Pol II phosphorylation at RBP1 initiates transcription  2. Transcription elongation Coding strand Template strand RNA Pol II is released from the promoter RNA Pol II unwinds the DNA into two separate strands– “transcription bubble” RNA Pol II moves along the DNA and uses one strand as a template to synthesise a complementary RNA sequence § Either of the strands can be a template – depends where the promoter is RNA Pol II adds nucleotides to 3’-end of the growing RNA molecule  3. Transcription termination RNA Pol II encounters a termination signal and transcription stops § Termination signal – AAUAAA hexamer in newly-formed mRNA RNA Pol and mRNA are released and the DNA double helix reforms A new cycle of transcription begins Protein Translation – Reading the code  Protein Translation The process of translation involves the conversion of genetic information from “RNA language” into “protein language” that uses different symbols  Codons There are only 4 different nucleotides in mRNA (A,U,G,C) but 20 different amino acids in proteins Not one-to-one correspondence Nucleotide sequence in an mRNA is read in groups of three called codons Each codon specifies one amino acid The code 64 possible codons (4 x 4 x 4) but only 20 amino acids Most amino acids are encoded by more than one codon – the code is redundant The codon for methionine (AUG) is also the start codon –translation initiation There are 3 stop codons – signal translation termination  Transfer RNAs (tRNAs) 3D shape Small RNA molecules called transfer (t)RNAs are made from a single strand of RNA folded into a cloverleaf shape (2D shape) § 80 nucleotides long The strand folds into a 2D complex 3D structure shape because base pairs form between nucleotides in different parts of the molecule. This creates double-stranded regions and loops, folding the tRNA into its shape.  Transfer RNAs (tRNAs) tRNAs act as adaptors translating the Amino acid mRNA code to the appropriate amino 3’ end acid Amino acid- 5’ end A region of the molecule called the accepting arm anticodon loop contains the anticodon Anticodon = three consecutive nucleotides that bind by base-pairing to the complementary codon on the mRNA Anticodon loop The amino acid attaches to a short single-stranded region at the 3’ end of Anticodon the molecule - amino acid-accepting arm  Coupling of amino acids to tRNAs Amino acid (Tryptophan) High-energy bond tRNA Amino acid is linked to tRNA Amino acyl-tRNA synthetase (Tryptophanyl-tRNA synthetase) The enzyme recognizes the amino acid, and the anticodon loop and the amino acid-accepting arm of the tRNA – matches the tRNA with the amino acid The enzyme links the amino acid to the tRNA using energy from ATP hydrolysis  Coupling of amino acids to tRNAs Recognition and attachment of each amino acid to the correct tRNA depends on the enzymes aminoacyl-tRNA synthetases § Catalyze the formation of a covalent bond between amino acid and tRNA § 20 synthetases, one for each amino acid The enzyme recognizes the nucleotides in the anticodon loop and the amino acid that will be attached to the amino acid-accepting arm of the tRNA The aminoacyl-tRNA synthetase-catalyzed reaction is coupled to hydrolysis of ATP § Produces a high energy bond linking the amino acid and tRNA The energy of this bond is later used to link the amino acid to the polypeptide chain  Wobble base-pairing and redundancy Both the CUC and CUU codons encode for leucine The two first nucleotides of the anticodon require accurate pairing The third nucleotide (G) can bind to either C or U – does not follow base- pairing rules Some amino acids are encoded by more than one codons - Redundancy This is because: 1. Some amino acids can bind to more than one tRNAs 2. Some tRNAs require accurate base-pairing only at two positions and can tolerate a mismatch at the third position – wobble base- pairing Protein Translation – Ribosomes  Ribosomes translate mRNA into protein mRNA is released from the nucleus into DNA the cytoplasm through nuclear pores DNA replication mRNA is attached to ribosomes, which translate it into proteins Transcription Free ribosomes translate proteins that mRNA mRNA Ø remain in the cytoplasm Ø are incorporated in mitochondria and mRNA nucleus Ribosome Endoplasmic reticulum ribosomes translate proteins that Ø are incorporated in the cell membrane Polypeptide Ø are destined to be released by the cell  Ribosomes Protein translation is carried out by ribosomes § Bind to the mRNA molecule and position the correct tRNA molecule § Catalyze formation of covalent bonds between amino acids to form a polypeptide chain – peptidyl transferase activity Ribosomes are large complexes made of small proteins (ribosomal proteins) and RNA (ribosomal RNAs) Composed of a large subunit and a small subunit which assemble to form a complete ribosome § Small subunit – matches tRNA to the mRNA codon § Large subunit – catalyzes peptide bonds between amino acids  Ribosomes structure 49 proteins + 3 rRNAs 33 proteins + 1 rRNA Large subunit Small Smallsubunit subunit 60S 40S 40S S units = sedimentation coefficient Large subunit Small subunit Complete ribosome (80S)  Ribosome structure Ribosomes consist of 2/3 RNA and 1/3 proteins by weight The RNA, and not the protein, is responsible for the catalytic activity and regulation of translation by the ribosome RNA is folded in a precise 3D structure that: 1. Forms the binding sites for the tRNAs 2. Forms the catalytic site of peptidyl transferase NOTE: RNA molecules can act as enzymes by forming active sites that allow the binding of a substrate and catalyze a biochemical reaction - ribozymes Proteins are involved in the folding and stabilization of the RNA structure  Ribosome function The small and large subunits assemble on an mRNA molecule near its 5’-end to start the synthesis of a protein The ribosome moves in a 5’ to 3’ direction translating the nucleotide sequence into an amino acid sequence, one codon at a time, using the tRNAs as adaptors Each amino acid is added in the correct sequence to the end of the polypeptide chain When protein synthesis is completed the two subunits separate Mechanism of protein translation  Translation initiation The tRNA recognizing the start codon (initiator tRNA) binds to the small ribosomal subunit and scans the mRNA until it finds the start codon (AUG) and binds to it The large subunit is then recruited to complete the assembly of the ribosome on the mRNA molecule near its 5’-end to start the synthesis of a protein  Translation elongation The ribosome moves in a 5’ to 3’ direction translating the nucleotide sequence into an amino acid sequence, one codon at a time, using the tRNAs as adaptors Each amino acid is added in the correct sequence to the end of the polypeptide chain  Translation termination The presence of a stop codon (UAA, UAG or UGA) signals protein synthesis termination When protein synthesis is completed the two subunits separate and the tRNAs and polypeptide is released  Ribosome binding sites mRNA-binding site § Site where the mRNA is bound to the ribosome Aminoacyl-tRNA (A) site § tRNA carrying an amino acid enters this site by base pairing with the complementary codon Peptidyl-tRNA (T) site § A tRNA carrying the growing mRNA-binding polypeptide is found in this site site Exit (E) site § The used tRNA enters the site to be ejected from the ribosome Mechanism of initiation The tRNA recognizing the start codon (AUG), called initiator tRNA, is loaded onto the P site of the small subunit together with proteins called translation initiation factors § Initiator tRNA always carries methionine The small subunit binds to the 5’ end of the mRNA and scans the sequence until it detects the AUG codon The anticodon of the initiator tRNA base pairs with the AUG codon Mechanism of initiation Recognition of the AUG codon by the initiator tRNA triggers the translation initiation factors to dissociate and the large subunit to bind and complete the ribosome assembly The tRNA carrying the next amino acid binds to the A site The large subunit catalyzes the uncoupling of methionine from the initiator tRNA and the formation of a peptide bond with the amino acid on the new tRNA in A site Mechanism of elongation A tRNA carrying the next amino acid to be added binds to the A site by forming base pairs with the mRNA codon § The tRNA carrying the growing polypeptide chain is in the P site The C-terminal of the polypeptide chain is uncoupled from the tRNA in the P site and joined by a peptide bond to the free N-terminal of the amino acid on the tRNA in the A site by the peptidyl transferase activity § The tRNA in the P site is now free Mechanism of elongation The large subunit moves in the 5’ to 3’ direction and the two tRNAs shift to the E and P sites § The free tRNA shifts to the E site § The tRNA linked to the polypeptide shifts to the P site The small subunit moves three nucleotides in the along the mRNA molecule in the 5’ to 3’ direction joining the large subunit and causing the free tRNA in the E site to be ejected § The A site is now free to receive a new tRNA Mechanism of termination Termination of translation is signalled by the presence of a stop codon (UAA, UAG or UGA) in the A site § Stop codons are not recognized by tRNAs and they do not specify an amino acid Proteins called release factors bind to the stop codons in the A site Mechanism of termination Binding of the release factors changes peptidyl transferase activity leading to the addition of a water molecule instead of an amino acid to the polypeptide chain The addition of the water causes the C- terminal of the polypeptide to be released from the tRNA (hydrolysis) Ribosome releases the mRNA and dissociates into its two subunits in order to assemble on another mRNA molecule