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Download Chapter 10 - Protein Synthesis: Transcription and Translation
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Protein Synthesis: Transcription and Translation aa aa From nucleus to cytoplasmaa aa aa aa aa transcription DNA mRNA aa aa protein aa translation trait nucleus cytoplasm Review Central Dogma of Molecular Biology DNA vs RNA Sugar: Bonds with Adenine: # of Strands: DNA RNA deoxyribose ribose thymine uracil two one Kinds of RNA • Messenger RNA (mRNA):copies DNA’s code & carries the genetic information to the ribosomes • Ribosomal RNA (rRNA):along with protein, makes up the ribosomes • Transfer RNA (tRNA): transfers amino acids to the ribosomes where proteins are synthesized Transcription • Making mRNA from DNA • ONE DNA strand is the template (pattern) – match bases • A: U • G:C • Enzyme – RNA polymerase Sense vs. Antisense Strand • The antisense strand is the template strand during transcription, which is copied for translation into a protein. • a sense strand, or coding strand, is the strand of DNA running from 5' to 3' that is complementary to the antisense strand of DNA, which runs from 3' to 5'. • The sense strand is the strand of DNA that has the same sequence as the mRNA. DNA contains codes Transcription in prokaryotes • RNA polymerase recognizes a specific base sequence in the DNA called a promoter and binds to it. • The promoter identifies the start of a gene, which strand is to be copied, and the direction that it is to be copied. RNA POLYMERASE- adds RNA nucleotides complimentary to the DNA template strand • Complimentary bases are assembled (U instead of T). • A termination code in the DNA indicates where transcription will stop in prokaryotes. • The mRNA produced is called a mRNA transcript. Transcription Eukaryotes • Step 1: Transcription factors proteins bind and double stranded DNA unzips as RNA polymerase binds to promoter (TATAA sequence “TATA box” or other promoter) T Matching bases of DNA & RNA • Step 2: Match RNA bases to DNA bases on one of the DNA strands. Built 5’ 3’, using DNA 3’-5’ template A G U A G G U U C A AG C C G A U A C A C C RNA polymerase A U G T G G T A C A G C T A G T C A T CG T A C CG T U C Matching bases of DNA & RNA • U instead of T is matched to A and the mRNA transcript is built 5’-3’ DNA mRNA TACGCACA TTTA CGTA CG AUGCGUGUAAAUGCAUGC Ending Transcription • Step 3: Transcription stops upon reading polyadenylation signal sequence. RNA Polymerase 5’ 3’ 15 mRNA Processing in Eukaryotes • After the DNA is transcribed into RNA, editing must be done to the nucleotide chain to make the RNA functional • Introns, non-functional segments of DNA are snipped out of the chain by snRNP’s (spliceosomes or ribonucleases) 16 mRNA Editing • Exons, segments of DNA that code for proteins, are then rejoined by the enzyme ligase • A guanine triphosphate cap is added to the 5” end of the newly copied mRNA • A poly A tail is added to the 3’ end of the RNA • The newly processed mRNA can then leave the nucleus through 17 nuclear pores Result of Transcription CAP New Transcript Tail 18 mRNA Transcript •mRNA leaves the nucleus through its pores and goes to the ribosomes 19 How does mRNA code for proteins • mRNA leaves nucleus • mRNA goes to ribosomes in cytoplasm • Proteins built from instructions on mRNA mRNA A C C A U G U C G A U C A GU A GC A U G GC A How? aa aa aa aa aa aa aa aa Translation mRNA to protein = Translation • The working instructions mRNA • The reader ribosome • The transporter transfer RNA (tRNA) ribosome mRNA A C C A U G U C G A U C A GU A GC A U G GC A U GG tRNA aa aa aa U A C tRNA aa A G tRNA aa C U AG tRNA aa Translation • Translation is the process of decoding the mRNA into a polypeptide chain • Ribosomes read mRNA three bases or 1 codon at a time and construct the proteins • The process begins by the mRNA leaving the nucleus through nuclear pores 23 Messenger RNA (mRNA) • Carries the information for a specific protein • Made up of 500 to 1000 nucleotides long • Sequence of 3 bases called codon • AUG – methionine or start codon • UAA, UAG, or UGA – stop codons Initiation and Termination Codes • An initiation code “start codon” signals the start of a genetic message. As the ribosome moves along a mRNA transcript, it will not begin synthesizing protein until it reaches an initiation code. (Ex. AUG) • Termination codes “stop codon” signal the end of the genetic message. Synthesis stops when the ribosome reaches a terminator codon.(Ex. UAA, UAG, UGA) mRNA codes for proteins in triplets DNA TACGCACATTTACGTACGCGG codon mRNA AUGCGUGUAAAUGCAUGC AUGCGUGUAAAUGCAUGCGCC Met protein Cys Ala Arg ribosome ? Val Asn Ala Codon = block of 3 mRNA bases The Genetic Code •Use the code by reading from the center to the outside •Example: AUG codes for Methionine •Genetic code contains a wobble at the third b.p.-there is redundancy in the genetic code, resulting in fewer errors. 27 Genetic Code- Another way Name the Amino Acids • • • • • GGG? UCA? CAU? GCA? AAA? 29 Translation overview Ribosomes • Made of a large and small subunit • Composed of rRNA (40%) and proteins (60%) • Have three sites for tRNA attachment --- A –accepting site and P site- peptide bond • The tRNA exits from the E site 31 Transfer RNA (tRNA) A C • Clover-leaf shape 3’ C 5’ • Single stranded molecule with attachment site at 3’ end for an amino acid • Opposite end has three nucleotide bases called the anticodon 32 Transfer RNA The amino acid is bound to the 3’ end by a covalent ester bond through a condensation reaction This is done by aminoacyl-tRNA synthetases or tRNA activating enzyme- one for every a.a. tRNA is held together by intramolecular base pairing There is a specific tRNA molecule for every codon or for every amino acid Codons and Anticodons • Recall a codon is made from 3 bases within mRNA • The 3 bases of an anticodon are complimentary to the 3 bases of a codon • Example: Codon ACU Anticodon UGA UGA ACU 34 The Process Step 1- Initiation • mRNA transcript start codon AUG attaches to the small ribosomal subunit • Small subunit attaches to large ribosomal subunit mRNA transcript 36 tRNA Binding Steps 1. The amino acid will react with ATP and become activated (with aid of enzyme ATPase). The ATP lose energy in this process 2. The activated amino acid will then bind to the acceptor stem (3’) of its own tRNA by a covalent bond aided by tRNA synthetase – The bond between the tRNA and the a.a is broken when the peptide bond is formed Step 2 - Elongation • As the ribosome moves, one tRNA carrying the Met amino acid moves into the A site, where the anticodon matches the mRNA transcript (AUG). • It then moves to the P site of the ribosome and another tRNA brings the next amino acid • Peptide bonds join the amino acids and leaves the P site tRNA empty (done by peptidyl transferase) • The empty tRNA is moved to the E site where it exits Ribosome “walks” the strand of mRNA 3 bases at a time 5’-3’ 1. Transfer of proper tRNA to A-site of ribosome. 2. peptidyl transfer/ formation of peptide bond 3. translocation Step 3-Termination • When the ribosome reads a stop codon. • When this code (UGA, UAA, UAG) is reached protein translation is terminated. • Release factor binds • Polypeptide dissociates from ribosome • Occurs once per protein End Product –The Protein! • The end products of protein synthesis is a primary structure of a protein • A sequence of amino acid bonded together by peptide bonds aa2 aa1 aa3 aa4 aa5 aa199 aa200 41 Ribosomes Large subunit E Site P Site A Site mRNA Small subunit A U G C U 42 Initiation aa2 aa1 2-tRNA G A U anticodon 1-tRNA U A C hydrogen bonds A U G codon C U A C mRNA 43 Elongation peptide bond aa1 aa3 aa2 3-tRNA 1-tRNA anticodon hydrogen bonds U A C A U G codon 2-tRNA G A A G A U C U A C U U C G A mRNA 44 aa1 peptide bond aa3 aa2 1-tRNA 3-tRNA U A C (leaves) 2-tRNA A U G G A A G A U C U A C U U C G A mRNA Ribosomes move over one codon 45 aa1 peptide bonds aa4 aa2 aa3 4-tRNA 2-tRNA A U G 3-tRNA G C U G A U G A A C U A C U U C G A A C U mRNA 46 aa1 peptide bonds aa4 aa2 aa3 2-tRNA 4-tRNA G A U (leaves) 3-tRNA A U G G C U G A A C U A C U U C G A A C U mRNA Ribosomes move over one codon 47 aa1 peptide bonds aa5 aa2 aa3 aa4 5-tRNA U G A 3-tRNA 4-tRNA G A A G C U G C U A C U U C G A A C U mRNA 48 peptide bonds aa1 aa5 aa2 aa3 aa4 5-tRNA U G A 3-tRNA G A A 4-tRNA G C U G C U A C U U C G A A C U mRNA Ribosomes move over one codon 49 aa4 aa5 Termination aa199 aa3 primary structure aa2 of a protein aa200 aa1 200-tRNA A C U terminator or stop codon C A U G U U U A G mRNA http://www.youtube.com/watch?v=5iS4CR PPDus 50 Mutation • A mutation is a change in amino acid sequence and can be either a; • Frameshift (deletion) Original DNA: THE BIG RED ANT ATE ONE FAT BUG Frameshift mutation: THB IGR EDA NTA TEO NEF ATB UG? • Point Mutation Original DNA: THE BIG RED ANT ATE ONE FAT BUG Point mutation: THA BIG RED ANT ATE ONE FAT BUG Silent, Missense, and Nonsense Mutations • Three kinds of point mutations can occur. A mutation that results in an amino acid substitution is called a missense mutation. • A mutation that results in a stop codon so that incomplete proteins are produced, it is called a nonsense mutation. • A mutation that produces a functioning protein is called a silent mutation. Terms • Splicesomes• removes introns and ligase joins • RNA polymerase• adds RNA nucleotides complimentary to the DNA template strand • Transcription factors• proteins that recognize specific sequences in DNA when making mRNA and help RNA polymerase bind • ATPase• converts ATP to ADP and releases energy to do work ( used to bond tRNA to mRNA and GTPase also used when adding a.a to tRNA) • tRNA synthetase (tRNA activating enzyme) • enzyme that catalyzes the esterification of a specific amino acid to a specific tRNA • Peptidyltransferase• forms peptide bonds and helps with protein release • recognizes tRNA code and adds specific a.a. (a covalent bond) • Protein release factors• Helps release peptide during termination of translation by recognizing the termination codon or stop codon in an mRNA sequence. Question: What would be the complimentary RNA strand for the following DNA sequence? DNA 5’-GCGTATG-3’ 54 Answer: • DNA 5’-GCGTATG-3’ • RNA 3’-CGCAUAC-5’ 55 Exercise - Understanding DNA, mRNA, tRNA, and protein. Template (anti-sense) strand Non-template strand GGG TAC mRNA tRNA Amino Acid CUU UCG Leu CCU