Ch. 17 Protein Synthesis BIOL 222

Transcription

1 Ch. 17 Protein Synthesis BIOL 222 The Flow of Gene3c Informa3on Central dogma of gene7cs One way flow of informa7on DNA mrna protein Informa7on in DNA is held in the specific sequences of nucleo7des DNA codes for specific proteins Links between genotype and phenotype Gene expression process by which DNA directs protein synthesis includes two stages: transcrip3on and transla3on Nutri3onal Mutants in Neurospora George Beadle and Edward Tatum Developed a one gene one enzyme hypothesis each gene dictates produc7on of a specific enzyme 1

2 The Products of Gene Expression one gene one protein Some proteins aren t enzymes Many proteins are composed of several polypep7des each of which has its own gene Therefore, Beadle and Tatum s hypothesis is now restated as the one gene one polypep0de hypothesis Note that it is common to refer to gene products as proteins rather than polypep7des Protein Synthesis Basic Principles of Transcrip3on and Transla3on Transcrip3on synthesis of RNA under the direc7on of DNA produces messenger RNA (mrna) Transla3on synthesis of a polypep7de Ribosomes sites of transla7on Basic Principles of Transcrip3on and Transla3on Prokaryotes mrna produced by transcrip7on is immediately translated without more processing Eukaryotes Nuclear envelope separates transcrip7on from transla7on Nuclear Envelope 2

3 Basic Principles of Transcrip3on and Transla3on Eukaryo7c RNA transcripts modified through RNA processing to yield finished mrna Primary transcript ini7al RNA transcript from any eukaryo7c gene The Gene3c Code Gene7c code System for reading sequence code in mrna 20 amino acids but only four nucleo7de bases in DNA Triplets of Bases Template strand (an3-sense) Only one of the two DNA strands acts as template for sequence of nucleo7des in RNA transcript Codon mrna base triplets read in the to direc7on during transla7on specifies the amino acid to be placed at the corresponding posi7on along a polypep7de 3

4 Codons along mrna molecule Read in the to direc7on Codons, Triplets of Bases DNA molecule Gene 1 Gene 2 Gene 3 DNA template strand TRANSCRIPTION mrna Codon TRANSLATION Protein Amino acid 64 codons Cracking the Code Deciphered by the mid-1960s 61 code for amino acids 3 are stop signals to end transla7on Redundancy of the gene7c code No codon specifies more than one amino acid Reading frame But one amino acid may be coded for by more than one codon Codons must be read in the correct groupings Fig Second mrna base First mrna base ( end of codon) Third mrna base ( end of codon) 4

5 Gene7c code Evolu3on of the Gene3c Code nearly universal shared by the simplest bacteria to the most complex animals Genes can be transcribed and translated azer being transplanted from one species to another (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene Molecular Components of Transcrip3on RNA polymerase Catalyzes RNA synthesis Adds new RNA nucleo7des follows the same base-pairing rules as DNA except uracil subs7tutes for thymine Molecular Components of Transcrip3on Promoter DNA sequence for RNA polymerase a[achment Terminator Sequence signaling the end of transcrip7on Transcrip3on unit The stretch of DNA that is transcribed 5

6 Fig Promoter Transcription unit DNA Start point RNA polymerase 1 Initiation Elongation Nontemplate strand of DNA Unwound DNA RNA transcript Template strand of DNA 2 Elongation RNA polymerase end RNA nucleotides Rewound DNA RNA transcript 3 Termination Newly made RNA Direction of transcription ( downstream ) Template strand of DNA Completed RNA transcript Synthesis of an RNA Transcript The three stages of transcrip7on: Ini7a7on Elonga7on Termina7on Transcrip3on factors mediate the binding of RNA polymerase and the ini7a7on of transcrip7on Transcrip3on ini3a3on complex The completed assembly of transcrip7on factors TATA box Ini3a3on of Transcrip3on and RNA polymerase II bound to a promoter A eukaryo7c promoter crucial in forming the ini7a7on complex 6

7 Fig A eukaryotic promoter includes a TATA box Promoter Template TATA box Start point Template DNA strand Transcription factors 2 Several transcription factors must bind to the DNA before RNA polymerase II can do so. RNA polymerase II 3 Additional transcription factors bind to the DNA along with RNA polymerase II, forming the transcription initiation complex. Transcription factors RNA transcript Transcription initiation complex Elonga3on RNA polymerase Travels down DNA Untwists the double helix 10 to 20 bases at a 7me Its own helicase ac7vity progresses at a rate of 40 nucleo7des per second in eukaryotes A gene can be transcribed simultaneously by several RNA polymerases Termina3on of Transcrip3on The mechanisms of termina7on are different in bacteria and eukaryotes Bacterial termina7on PLAY polymerase stops transcrip7on at the end of the terminator Eukaryo7c termina7on RNA Polymerase con7nues through the polyadenyla7on (polya) signal TTATTT (transcribed to AAUAAA) Con7nues transcrip7on azer the pre-mrna is cleaved from the growing RNA chain AZer nucleo7des, polymerase eventually falls off the DNA 7

8 Altera3on of mrna Ends Each end of a pre-mrna molecule is modified in a par7cular way: The end receives a modified nucleo7de cap Methylated guanine nucleo7de The end gets a poly-a tail ~ Provide several func7ons: They seem to facilitate the export of mrna They protect mrna from hydroly7c enzymes They help ribosomes a[ach to the end Protein-coding segment Polyadenylation signal AAA G P P P AAUAAA AAA Cap UTR Start codon Stop codon UTR Poly-A tail Split Genes and RNA Splicing Primary Transcript Ini7al eukaryo7c RNA before processing Introns Noncoding regions of primary transcript Exons Regions of transcript translated into amino acid sequences RNA splicing Removes introns and joins exons together crea7ng an mrna molecule with a con7nuous coding sequence Fig Exon Intron Exon Intron Exon Pre-mRNA Cap Poly-A tail Coding segment Introns cut out and exons spliced together mrna Cap Poly-A tail UTR UTR PLAY 8

9 RNA Processing RNA splicing is carried out by spliceosomes Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snrnps) recognize the splice sites short sequences of nucleo7des at the end of each intron Fig RNA transcript (pre-mrna) Exon 1 Intron Exon 2 Protein snrna snrnps Other proteins Fig RNA transcript (pre-mrna) Exon 1 Intron Exon 2 Protein snrna snrnps Other proteins Spliceosome 9

10 Fig RNA transcript (pre-mrna) Exon 1 Intron Exon 2 Protein snrna snrnps Other proteins Spliceosome PLAY Spliceosome components mrna Exon 1 Exon 2 Cut-out intron Ribozymes Ribozymes cataly7c RNA molecules func7on as enzymes and can splice RNA Showed that not all biological catalysts were proteins Ribozymes Three proper7es of RNA enable it to func7on as an enzyme It can form a three-dimensional structure because of its ability to base pair with itself Some bases in RNA contain func7onal groups Amine, carbonyl RNA may hydrogen-bond with other nucleic acid molecules 10

11 Alterna3ve RNA splicing Alterna3ve Splicing Some genes can encode more than one kind of polypep7de depending on which segments are treated as exons during RNA splicing Therefore the number of polypep7des an organism can produce is greater than its number of genes Humans: ~25,000 genes ~150,000 proteins Alterna3ve Splicing Domains Regions of proteins with discrete modular architecture Different exons code for the different domains in a protein Exon shuffling may result in the evolu7on of new proteins Fig DNA Gene Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide 11

12 Transla3on Process of reading mrna to guide construc7on of specific polypep7de Occurs in cytoplasm At a ribosome Using transfer RNA (trna) To deliver amino acids to ribosome Transfer RNA Transfer RNA (trna) serves as a bridging molecule in protein synthesis ability to bind with both a specific amino acid and mrna Delivers specific amino acid to a ribosome in which an mrna transcript is being read. RNA molecule ~80nt long folded back on itself ~45 types per cell due to trna wobble all have CCA at 3 end recogni7on for Addi7on of amino acid to trna catalyzed by aminoacyl-trna synthetase 20 different types one for each amino acid trna Structure/Charging 12

13 trna Structure An7codon Opposite end of the trna molecule from the amino acid binds with the complimentary codon in the mrna chain Therefore (almost) iden7cal to the original DNA template! Ribosome workbenches of protein synthesis composed of proteins and ribosomal RNA (rrna, ~60%) Enzyma7c RNA (performs catalysis) Protein for structure Each ribosome exists as two separate subunits in the cytoplasm come together only with the ini7a7on of protein transla7on. Small subunit Ribosome Structure mrna binding Large subunit Three trna binding sites Aminoacyl (A site) trna entry site Pep7dyl (P site) Polypep7de transfer site Exit (E site) 13

14 Three Stages of Transla3on Chain Ini7a7on Brings together mrna, small subunit, first trna, and finally, large subunit Chain Elonga7on Addi7on of amino acids Chain Termina7on Signals end of polypep7de and stops transla7on Transla3on Ini3a3on mrna binds with small ribosomal subunit At recogni7on sequence and methylated cap on 5 end Upstream from start (ini7a7on) codon First trna binds to mrna start codon AUG trna an7codon UAC Codes for methionine Transla3on Ini3a3on Large subunit binds GTP powered Met trna now in Pep7dyl site Aminoacyl site ready for next trna and elonga7on 14

15 Transla3on Elonga3on Succession of trna molecules arriving at a ribosome Dictated by an7codon-codon complementa7on Facilitates delivery of specific amino acids Transla3on Elonga3on (cont.) Step 1 - Codon Recogni3on Elonga7on Factor guides trna into A site Requires hydrolysis of more GTP Hydrogen bonds form base pairs between codon-an7codon Transla3on Elonga3on (cont.) Step 2 - Pep3de bond forma3on Between first Met amino acid in P site and next amino acid in A site Catalyzed by pep7dyl transferase Part of large subunit Met detachment From Met trna Growing polypep7de now a[ached to trna in A site 15

16 Transla3on Elonga3on (cont.) Step 3 - Transloca3on mrna ratcheted one codon from 5-3 Uses GTP Moves (empty) Met trna to E site Second trna now in P site Next codon now in A site ready for next trna Empty Met trna exits Back to step 1 codon recogni7on In A site Transla3on Termina3on Step 1 - Termina7on (stop) mrna codon enters A site UAA, UAG, UGA Release Factor binds stop codon Step 2 - Polypep7de release Transla3on Termina3on Pep7dyl transferase adds water molecule instead of amino acid at stop codon Caused by release factor H 2 0 hydrolyzes polypep7de from trna in P site 16

17 Transla3on Termina3on Step 3 - Ribosome Dissocia7on Small and large subunits separate Polypep7de now ready to fold May have begun already Transla3on Translation Polyribosomes Mul7ple ribosomes translate single mrna Increases rate of protein produc7on for the cell Also called polysomes 17

18 Protein folding Postransla3onal Modifica3ons Primary structure occurs as polypep7de formed Secondary, Ter7ary subsequent May be facilitated by chaperonin Further modifica7ons Sugars, Lipids, Phosphates, etc Targe7ng to loca7on Signal pep3de First ~20 amino acids Triggers delivery to ER Detected by signal recogni7on par7cle (SRP) You should now be able to: Describe the contribu7ons made by Beadle, and Tatum to our understanding of the rela7onship between genes and enzymes Briefly explain how informa7on flows from gene to protein Compare transcrip7on and transla7on in bacteria and eukaryotes Explain what it means to say that the gene7c code is redundant and unambiguous Include the following terms in a descrip7on of transcrip7on: mrna, RNA polymerase, the promoter, the terminator, the transcrip7on unit, ini7a7on, elonga7on, termina7on, and introns Include the following terms in a descrip7on of transla7on: trna, wobble, ribosomes, ini7a7on, elonga7on, and termina7on 18