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Lecture 22 Protein synthesis 2 Initiation of Translation • A leader sequence on mRNA attaches to the small subunit of the ribosome • Methionine-charged initiator tRNA binds to the small subunit • The large ribosomal unit now binds to this complex forming a functional ribosome 10.8 The genetic code is the Rosetta stone of life – Characteristics of the genetic code – Triplet: Three nucleotides specify one amino acid – 61 codons correspond to amino acids – AUG codes for methionine and signals the start of transcription – 3 “stop” codons signal the end of translation Copyright © 2009 Pearson Education, Inc. 10.8 The genetic code is the Rosetta stone of life – Redundant: More than one codon for some amino acids – Unambiguous: Any codon for one amino acid does not code for any other amino acid – Does not contain spacers or punctuation: Codons are adjacent to each other with no gaps in between – Nearly universal Copyright © 2009 Pearson Education, Inc. Genetic Code • RNA codons code for amino acids according to a genetic code Figure 3.35 Strand to be transcribed DNA Strand to be transcribed DNA Transcription RNA Start codon Stop codon Strand to be transcribed DNA Transcription RNA Start codon Polypeptide Met Translation Lys Phe Stop codon • Codon = – Determines the insertion of a specific amino acid during protein synthesis – GCU ACG GAG Alanine Threonine Glutamic Acid – Or the signal to stop protein synthesis 10.10 Eukaryotic RNA is processed before leaving the nucleus – Messenger RNA (mRNA) contains codons for protein sequences – Eukaryotic mRNA has interrupting sequences called introns, separating the coding regions called exons – Eukaryotic mRNA undergoes processing before leaving the nucleus – Cap added to 5’ end: single guanine nucleotide – Tail added to 3’ end: Poly-A tail of 50–250 adenines – RNA splicing: removal of introns and joining of exons to produce a continuous coding sequence Copyright © 2009 Pearson Education, Inc. Exon Intron Exon Intron Exon DNA Cap RNA transcript with cap and tail Transcription Addition of cap and tail Introns removed Tail Exons spliced together mRNA Coding sequence Nucleus Cytoplasm 10.11 Transfer RNA molecules serve as interpreters during translation – Transfer RNA (tRNA) molecules match an amino acid to its corresponding mRNA codon – tRNA structure allows it to convert one language to the other – An amino acid attachment site allows each tRNA to carry a specific amino acid – An anticodon allows the tRNA to bind to a specific mRNA codon, complementary in sequence – A pairs with U, G pairs with C Copyright © 2009 Pearson Education, Inc. tRNA • Anticodon matches to mRNA codon and allows the amino acid to be placed in a location for bonding to the growing amino acid chain Animo Acid Amino acid attachment site Hydrogen bond RNA polynucleotide chain Anticodon 10.12 Ribosomes build polypeptides – Translation occurs on the surface of the ribosome – – – – Ribosomes have two subunits: small and large Each subunit is composed of ribosomal RNAs and proteins Ribosomal subunits come together during translation Ribosomes have binding sites for mRNA and tRNAs Copyright © 2009 Pearson Education, Inc. tRNA molecules Growing polypeptide Large subunit mRNA Small subunit tRNA-binding sites Large subunit mRNA binding site Small subunit Next amino acid to be added to polypeptide Growing polypeptide tRNA mRNA Codons 10.13 An initiation codon marks the start of an mRNA message – Initiation brings together the components needed to begin RNA synthesis – Initiation occurs in two steps 1. mRNA binds to a small ribosomal subunit, and the first tRNA binds to mRNA at the start codon – The start codon reads AUG and codes for methionine – The first tRNA has the anticodon UAC 2. A large ribosomal subunit joins the small subunit, allowing the ribosome to function – The first tRNA occupies the P site, which will hold the growing peptide chain – The A site is available to receive the next tRNA Copyright © 2009 Pearson Education, Inc. Start of genetic message End Large ribosomal subunit Initiator tRNA P site 1 mRNA Start codon Small ribosomal subunit 2 A site 10.14 Elongation adds amino acids to the polypeptide chain until a stop codon terminates translation – Elongation is the addition of amino acids to the polypeptide chain – Each cycle of elongation has three steps 1. Codon recognition: next tRNA binds to the mRNA at the A site 2. Peptide bond formation: joining of the new amino acid to the chain – Amino acids on the tRNA at the P site are attached by a covalent bond to the amino acid on the tRNA at the A site Copyright © 2009 Pearson Education, Inc. 10.14 Elongation adds amino acids to the polypeptide chain until a stop codon terminates translation 3. Translocation: tRNA is released from the P site and the ribosome moves tRNA from the A site into the P site Copyright © 2009 Pearson Education, Inc. 10.14 Elongation adds amino acids to the polypeptide chain until a stop codon terminates translation – Elongation continues until the ribosome reaches a stop codon – Applying Your Knowledge How many cycles of elongation are required to produce a protein with 100 amino acids? – Termination – The completed polypeptide is released – The ribosomal subunits separate – mRNA is released and can be translated again Copyright © 2009 Pearson Education, Inc. Amino acid Polypeptide A site P site Anticodon mRNA Codons 1 Codon recognition Amino acid Polypeptide A site P site Anticodon mRNA Codons 1 Codon recognition 2 Peptide bond formation Amino acid Polypeptide A site P site Anticodon mRNA Codons 1 Codon recognition 2 Peptide bond formation New peptide bond 3 Translocation Amino acid Polypeptide A site P site Anticodon mRNA Codons 1 Codon recognition mRNA movement Stop codon 2 Peptide bond formation New peptide bond 3 Translocation 10.15 Review: The flow of genetic information in the cell is DNA RNA protein – Does translation represent: – DNA RNA or RNA protein? – Where does the information for producing a protein originate: – DNA or RNA? – Which one has a linear sequence of codons: – rRNA, mRNA, or tRNA? – Which one directly influences the phenotype: – DNA, RNA, or protein? Copyright © 2009 Pearson Education, Inc. Transcription DNA mRNA 1 mRNA is transcribed from a DNA template. RNA polymerase Translation Amino acid 2 Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Enzyme ATP tRNA Anticodon Large ribosomal subunit Initiator tRNA Start Codon mRNA 3 Initiation of polypeptide synthesis The mRNA, the first tRNA, and the ribosomal sub-units come together. Small ribosomal subunit New peptide bond forming Growing polypeptide Codons mRNA 4 Elongation A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome, one codon at a time. Polypeptide Stop codon 5 Termination The ribosome recognizes a stop codon. The polypeptide is terminated and released. Transcription DNA mRNA RNA polymerase 1 mRNA is transcribed from a DNA template. Translation Amino acid 2 Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Enzyme ATP tRNA Anticodon Large ribosomal subunit Initiator tRNA 3 Initiation of polypeptide synthesis The mRNA, the first tRNA, and the ribosomal sub-units come together. Start Codon mRNA Small ribosomal subunit New peptide bond forming Growing polypeptide 4 Elongation Codons A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome, one codon at a time. mRNA Polypeptide 5 Termination Stop codon The ribosome recognizes a stop codon. The polypeptide is terminated and released. 10.16 Mutations can change the meaning of genes – A mutation is a change in the nucleotide sequence of DNA – Base substitutions: replacement of one nucleotide with another – Effect depends on whether there is an amino acid change that alters the function of the protein – Deletions or insertions – Alter the reading frame of the mRNA, so that nucleotides are grouped into different codons – Lead to significant changes in amino acid sequence downstream of mutation – Cause a nonfunctional polypeptide to be produced Copyright © 2009 Pearson Education, Inc. 10.16 Mutations can change the meaning of genes – Mutations can be – Spontaneous: due to errors in DNA replication or recombination – Induced by mutagens – High-energy radiation – Chemicals Copyright © 2009 Pearson Education, Inc. Normal hemoglobin DNA Mutant hemoglobin DNA mRNA mRNA Normal hemoglobin Glu Sickle-cell hemoglobin Val Normal gene mRNA Protein Met Lys Phe Gly Ala Lys Phe Ser Ala Base substitution Met Base deletion Missing Met Lys Leu Ala His Nucleus Nuclear membrane RNA polymerase Nuclear pore mRNA Template strand of DNA Amino acids Released mRNA 1 After mRNA processing, mRNA leaves nucleus and attaches to ribosome, and translation begins. tRNA Aminoacyl-tRNA synthetase Small ribosomal subunit Codon 15 Codon 16 Codon 17 Direction of ribosome advance Portion of mRNA already translated tRNA “head” bearing anticodon Large ribosomal subunit 2 4 Once its amino acid is released, tRNA is ratcheted to the E site and then released to reenter the cytoplasmic pool, ready to be recharged with a new amino acid. 3 As the ribosome moves along the mRNA, a new amino acid is added to the growing protein chain and the tRNA in the A site is translocated to the P site. Incoming aminoacyltRNA hydrogen bonds via its anticodon to complementary mRNA sequence (codon) at the A site on the ribosome. Energized by ATP, the correct amino acid is attached to each species of tRNA by aminoacyl-tRNA synthetase enzyme. Figure 3.36 Nucleus Nuclear membrane RNA polymerase Nuclear pore mRNA Template strand of DNA Released mRNA Figure 3.36 Nucleus Nuclear membrane RNA polymerase Nuclear pore mRNA Template strand of DNA Released mRNA 1 After mRNA processing, mRNA leaves nucleus and attaches to ribosome, and translation begins. Small ribosomal subunit Codon 15 Codon 16 Codon 17 Direction of ribosome advance Portion of mRNA already translated Large ribosomal subunit Figure 3.36 Nucleus Nuclear membrane RNA polymerase Nuclear pore mRNA Template strand of DNA Amino acids Released mRNA 1 After mRNA processing, mRNA leaves nucleus and attaches to ribosome, and translation begins. Aminoacyl-tRNA synthetase Small ribosomal subunit Codon 15 Codon 16 Codon 17 tRNA Direction of ribosome advance Portion of mRNA already translated Large ribosomal subunit Energized by ATP, the correct amino acid is attached to each species of tRNA by aminoacyl-tRNA synthetase enzyme. Figure 3.36 Nucleus Nuclear membrane RNA polymerase Nuclear pore mRNA Template strand of DNA Amino acids Released mRNA 1 After mRNA processing, mRNA leaves nucleus and attaches to ribosome, and translation begins. tRNA Aminoacyl-tRNA synthetase Small ribosomal subunit Codon 15 Codon 16 Codon 17 Direction of ribosome advance Portion of mRNA already translated tRNA “head” bearing anticodon Large ribosomal subunit 2 Incoming aminoacyltRNA hydrogen bonds via its anticodon to complementary mRNA sequence (codon) at the A site on the ribosome. Energized by ATP, the correct amino acid is attached to each species of tRNA by aminoacyl-tRNA synthetase enzyme. Figure 3.36 Nucleus Nuclear membrane RNA polymerase Nuclear pore mRNA Template strand of DNA Amino acids Released mRNA 1 After mRNA processing, mRNA leaves nucleus and attaches to ribosome, and translation begins. tRNA Aminoacyl-tRNA synthetase Small ribosomal subunit Codon 15 Codon 16 Codon 17 Direction of ribosome advance Portion of mRNA already translated tRNA “head” bearing anticodon Large ribosomal subunit 2 3 As the ribosome moves along the mRNA, a new amino acid is added to the growing protein chain and the tRNA in the A site is translocated to the P site. Incoming aminoacyltRNA hydrogen bonds via its anticodon to complementary mRNA sequence (codon) at the A site on the ribosome. Energized by ATP, the correct amino acid is attached to each species of tRNA by aminoacyl-tRNA synthetase enzyme. Figure 3.36 Nucleus Nuclear membrane RNA polymerase Nuclear pore mRNA Template strand of DNA Amino acids Released mRNA 1 After mRNA processing, mRNA leaves nucleus and attaches to ribosome, and translation begins. tRNA Aminoacyl-tRNA synthetase Small ribosomal subunit Codon 15 Codon 16 Codon 17 Direction of ribosome advance Portion of mRNA already translated tRNA “head” bearing anticodon Large ribosomal subunit 2 4 Once its amino acid is released, tRNA is ratcheted to the E site and then released to reenter the cytoplasmic pool, ready to be recharged with a new amino acid. 3 As the ribosome moves along the mRNA, a new amino acid is added to the growing protein chain and the tRNA in the A site is translocated to the P site. Incoming aminoacyltRNA hydrogen bonds via its anticodon to complementary mRNA sequence (codon) at the A site on the ribosome. Energized by ATP, the correct amino acid is attached to each species of tRNA by aminoacyl-tRNA synthetase enzyme. Figure 3.36 Information Transfer from DNA to RNA • DNA triplets are transcribed into mRNA codons by RNA polymerase • Codons base pair with tRNA anticodons at the ribosomes • Amino acids are peptide bonded at the ribosomes to form polypeptide chains • Start and stop codons are used in initiating and ending translation Information Transfer from DNA to RNA Figure 3.38 Other Roles of RNA • Antisense RNA – prevents protein-coding RNA from being translated • MicroRNA – small RNAs that interfere with mRNAs made by certain exons • Riboswitches – mRNAs that act as switches regulating protein synthesis in response to environmental conditions Animation: http://www.youtube.com/watch ?v=NJxobgkPEAo http://www.youtube.com/watch?v=D3fOXt4 MrOM&feature=related Thank you