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The Molecular Biology of Genes and Gene Expression Central Dogma • First described by Francis Crick • Information only flows from DNA → RNA → protein • Transcription = DNA → RNA • Translation = RNA → protein • Retroviruses violate this order using reverse transcriptase to convert their RNA genome into DNA 2 How Genes Work: A Primer replication (mutation!) “software” ~ DNA, RNA genes DNA (nucleotides: A,T,G,C) transcription RNA (nucleotides: A,U,G,C) translation Protein (amino acids) “hardware” ~ proteins The Nature of Genes • Early ideas to explain how genes work came from studying human diseases • Archibald Garrod – 1902 – Recognized that alkaptonuria is inherited via a recessive allele – Proposed that patients with the disease lacked a particular enzyme • These ideas connected genes to enzymes 4 Beadle and Tatum – 1941 • Deliberately set out to create mutations in chromosomes and verify that they behaved in a Mendelian fashion in crosses • Studied Neurospora crassa – Used X-rays to damage DNA – Looked for nutritional mutations • Had to have minimal media supplemented to grow 5 • Beadle and Tatum looked for fungal cells lacking specific enzymes – The enzymes were required for the biochemical pathway producing the amino acid arginine – They identified mutants deficient in each enzyme of the pathway • One-gene/one-enzyme hypothesis has been modified to one-gene/one-polypeptide hypothesis 6 7 8 • Transcription – – – – DNA-directed synthesis of RNA Only template strand of DNA used U (uracil) in DNA replaced by T (thymine) in RNA mRNA used to direct synthesis of polypeptides • Translation – Synthesis of polypeptides – Takes place at ribosome – Requires several kinds of RNA 9 RNA • All synthesized from DNA template by transcription • Messenger RNA (mRNA) • Ribosomal RNA (rRNA) • Transfer RNA (tRNA) • Small nuclear RNA (snRNA) • Signal recognition particle RNA • Micro-RNA (miRNA) 10 Genetic Code • Francis Crick and Sydney Brenner determined how the order of nucleotides in DNA encoded amino acid order • Codon – block of 3 DNA nucleotides corresponding to an amino acid • Introduced single nulcleotide insertions or deletions and looked for mutations – Frameshift mutations • Indicates importance of reading frame 11 12 • Marshall Nirenberg identified the codons that specify each amino acid • Stop codons – 3 codons (UUA, UGA, UAG) used to terminate translation • Start codon – Codon (AUG) used to signify the start of translation • Code is degenerate, meaning that some amino acids are specified by more than one codon 13 14 Code practically universal • Strongest evidence that all living things share common ancestry • Advances in genetic engineering • Mitochondria and chloroplasts have some differences in “stop” signals 15 Prokaryotic transcription • Single RNA polymerase • Initiation of mRNA synthesis does not require a primer • Requires – Promoter – Start site – Termination site Transcription unit 16 • Promoter – Forms a recognition and binding site for the RNA polymerase – Found upstream of the start site – Not transcribed – Asymmetrical – indicate site of initiation and direction of transcription 17 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Core enzyme 9 TATAAT– Promoter (–10 sequence) Holoenzyme 5׳ 3׳ Downstream Start site (+1) TTGACA–Promoter (–35 sequence) Template strand Coding strand Prokaryotic RNA polymerase Upstream 5 ׳3׳ b. a. binds to DNA RNA polymerase bound to unwound DNA Transcription bubble 5׳ 3׳ dissociates ATP Helix opens at –10 sequence Start site RNA synthesis begins 5 ׳3׳ 18 • Elongation – Grows in the 5′-to-3′ direction as ribonucleotides are added – Transcription bubble – contains RNA polymerase, DNA template, and growing RNA transcript – After the transcription bubble passes, the now-transcribed DNA is rewound as it leaves the bubble 19 20 • Termination – Marked by sequence that signals “stop” to polymerase • Causes the formation of phosphodiester bonds to cease • RNA–DNA hybrid within the transcription bubble dissociates • RNA polymerase releases the DNA • DNA rewinds – Hairpin 21 22 • Prokaryotic transcription is coupled to translation – mRNA begins to be translated before transcription is finished – Operon • Grouping of functionally related genes • Multiple enzymes for a pathway • Can be regulated together 23 24 Eukaryotic Transcription • 3 different RNA polymerases – RNA polymerase I transcribes rRNA – RNA polymerase II transcribes mRNA and some snRNA – RNA polymerase III transcribes tRNA and some other small RNAs • Each RNA polymerase recognizes its own promoter 25 • Initiation of transcription – Requires a series of transcription factors • Necessary to get the RNA polymerase II enzyme to a promoter and to initiate gene expression • Interact with RNA polymerase to form initiation complex at promoter • Termination – Termination sites not as well defined 26 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Other transcription factors RNA polymerase II Eukaryotic DNA Transcription factor TATA box Initiation complex 27 Enhancers determine the temporal and spatial transcription patterns of genes (drawing modified from Tijan, R., Molecular Machines That Control Genes, Scientific American 272, Feb, 1995) Enhancers Activators = + Auxiliary Transcription Factors - Repressor = auxiliary transcription factor Silencer REPRESSOR Basal Transcription Factors H E F B A Co-activators TATA BOX RNA Polymerase CORE PROMOTER transcription mRNA modifications • In eukaryotes, the primary transcript must be modified to become mature mRNA – Addition of a 5′ cap • Protects from degradation; involved in translation initiation – Addition of a 3′ poly-A tail • Created by poly-A polymerase; protection from degradation – Removal of non-coding sequences (introns) • Pre-mRNA splicing done by spliceosome 29 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5´ cap HO OH P P CH2 P + 3´ N+ CH3 Methyl group P 5´ P P mRNA CH3 30 Eukaryotic pre-mRNA splicing • Introns – non-coding sequences • Exons – sequences that will be translated • Small ribonucleoprotein particles (snRNPs) recognize the intron–exon boundaries • snRNPs cluster with other proteins to form spliceosome – Responsible for removing introns 31 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. E1 I1 E2 I2 E3 I3 DNA template Transcription E4 I4 Exons Introns 5 ׳cap 33 ׳poly-A tail Primary RNA transcript Introns are removed 5 ׳cap a. 3 ׳poly-A tail Mature mRNA 32 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. E1 I1 E2 I2 E3 I3 DNA template E4 I4 Exons Introns Transcription 5 ׳cap 3 ׳poly-A tail Primary RNA transcript Introns are removed 3 ׳poly-A tail 5 ׳cap a. Mature mRNA Intron 1 mRNA 3 2 4 DNA 7 5 6 Exon b. c. b: Courtesy of Dr. Bert O’Malley, Baylor College of Medicine 33 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. snRNA Exon 1 snRNPs Intron Exon 2 A 5´ 3´ Branch point A 1. snRNA forms base-pairs with 5´ end of intron, and at branch site. 34 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. snRNA Exon 1 snRNPs Exon 2 Intron A 3´ 5´ Branch point A 1. snRNA forms base-pairs with 5´ end of intron, and at branch site. Spliceosome A 5´ 3´ 2. snRNPs associate with other factors to form spliceosome. 35 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. snRNA Exon 1 snRNPs Exon 2 Intron A 3´ 5´ Branch point A 1. snRNA forms base-pairs with 5´ end of intron, and at branch site. Spliceosome A 5´ 3´ 2. snRNPs associate with other factors to form spliceosome. Lariat A 5´ 3´ 3. 5´ end of intron is removed and forms bond at branch site, forming a lariat. The 3´ end of the intron is then cut. 36 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. snRNA Exon 1 snRNPs Intron Exon 2 A 5´ 3´ Branch point A 1. snRNA forms base-pairs with 5´ end of intron, and at branch site. Spliceosome A 5´ 3´ 2. snRNPs associate with other factors to form spliceosome. Lariat A 5´ 3´ 3. 5´ end of intron is removed and forms bond at branch site, forming a lariat. The 3´ end of the intron is then cut. Exon 1 5´ Excised intron Exon 2 Mature mRNA 3´ 4. Exons are joined; spliceosome disassembles. 37 Alternative splicing • Single primary transcript can be spliced into different mRNAs by the inclusion of different sets of exons • 15% of known human genetic disorders are due to altered splicing • 35 to 59% of human genes exhibit some form of alternative splicing • Explains how 25,000 genes of the human genome can encode the more than 80,000 different mRNAs 38 5’ ut exon 1 DNA intron 1 exon 2 intron 2 3’ ut exon 3 hnRNA RNA splicing mRNA 1 RNA splicing translation mRNA 2 translation protein 1 Differential RNA splicing can lead to different protein products protein 2 tRNA and Ribosomes • tRNA molecules carry amino acids to the ribosome for incorporation into a polypeptide – Aminoacyl-tRNA synthetases add amino acids to the acceptor stem of tRNA – Anticodon loop contains 3 nucleotides complementary to mRNA codons 40 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2D “Cloverleaf” Model 3D Ribbon-like Model Acceptor end Acceptor end 3׳ 5׳ Anticodon loop 3D Space-filled Model Acceptor end Anticodon loop Anticodon loop Icon Acceptor end Anticodon end c: Created by John Beaver using ProteinWorkshop, a product of the RCSB PDB, and built using the Molecular Biology Toolkit developed by John Moreland and Apostol Gramada (mbt.sdsc.edu). The MBT is fi nanced by grant GM63208 41 tRNA charging reaction • Each aminoacyl-tRNA synthetase recognizes only 1 amino acid but several tRNAs • Charged tRNA – has an amino acid added using the energy from ATP – Can undergo peptide bond formation without additional energy • Ribosomes do not verify amino acid attached to tRNA 42 43 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Amino group NH3+ ATP Pi Pi Carboxyl group Trp C O O– Amino acid site tRNA site Aminoacyl-tRNA synthetase 44 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Amino group NH3+ ATP Pi Pi tRNA site Carboxyl group Trp C O O– Amino acid site Accepting site OH tRNA Aminoacyl-tRNA Anticodon synthetase specific to tryptophan 45 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Amino group NH3+ ATP Pi Pi tRNA site Carboxyl group Trp C Charged tRNA travels to ribosome O O– Amino acid site NH3+ Trp Accepting site C O O OH tRNA Aminoacyl-tRNA Anticodon synthetase specific to tryptophan Charged tRNA dissociates 46 • The ribosome has multiple tRNA binding sites – P site – binds the tRNA attached to the growing peptide chain – A site – binds the tRNA carrying the next amino acid – E site – binds the tRNA that carried the last amino acid 47 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Large subunit 3´ Small subunit Large subunit 90° Small subunit Large subunit 0° mRNA Small subunit 5´ 48 • The ribosome has two primary functions – Decode the mRNA – Form peptide bonds • Peptidyl transferase – Enzymatic component of the ribosome – Forms peptide bonds between amino acids 49 Translation • In prokaryotes, initiation complex includes – Initiator tRNA charged with N-formylmethionine – Small ribosomal subunit – mRNA strand • Ribosome binding sequence (RBS) of mRNA positions small subunit correctly • Large subunit now added • Initiator tRNA bound to P site with A site empty 50 51 • Initiations in eukaryotes similar except – Initiating amino acid is methionine – More complicated initiation complex – Lack of an RBS – small subunit binds to 5′ cap of mRNA 52 • Elongation adds amino acids – 2nd charged tRNA can bind to empty A site – Requires elongation factor called EF-Tu to bind to tRNA and GTP – Peptide bond can then form – Addition of successive amino acids occurs as a cycle 53 54 55 • There are fewer tRNAs than codons • Wobble pairing allows less stringent pairing between the 3′ base of the codon and the 5′ base of the anticodon • This allows fewer tRNAs to accommodate all codons 56 • Termination – Elongation continues until the ribosome encounters a stop codon – Stop codons are recognized by release factors which release the polypeptide from the ribosome 57 58 Protein targeting • In eukaryotes, translation may occur in the cytoplasm or the rough endoplasmic reticulum (RER) • Signal sequences at the beginning of the polypeptide sequence bind to the signal recognition particle (SRP) • The signal sequence and SRP are recognized by RER receptor proteins • Docking holds ribosome to RER • Beginning of the protein-trafficking pathway 59 60 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA RNA polymerase polymerase IIII 1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript. 3´ 5´ Primary Primary RNA RNA transcript transcript 61 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA RNA polymerase polymerase IIII 1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript. 3´ Primary Primary RNA RNA transcript transcript 5´ Primary Primary RNA RNA transcript transcript 2. The primary transcript is processed by addition of a 5´ methyl-G cap, cleavage and polyadenylation of the 3´ end, and removal of introns. The mature mRNA is then exported through nuclear pores to the cytoplasm. Poly-A tail tail Poly-A Cut intron intron Cut Mature Mature mRNA mRNA 5´ cap cap 5´ 62 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA RNA polymerase polymerase IIII 1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript. 3´ Primary RNA transcript 5´ Primary Primary RNA RNA transcript transcript 2. The primary transcript is processed by addition of a 5´ methyl-G cap, cleavage and polyadenylation of the 3´ end, and removal of introns. The mature mRNA is then exported through nuclear pores to the cytoplasm. Poly-A tail Cut intron Mature mRNA 5´ cap 3. The 5´ cap of the mRNA associates with the small subunit of the ribosome. 5´ cap The initiator tRNA and large subunit are added to form an initiation complex. Large subunit mRNA Small subunit Cytoplasm 63 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA RNA polymerase polymerase IIII 1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript. 3´ 5´ Primary Primary RNA RNA transcript transcript 2. The primary transcript is processed by addition of a 5´ methyl-G cap, cleavage and polyadenylation of the 3´ end, and removal of introns. The mature mRNA is then exported through nuclear pores to the cytoplasm. Primary RNA transcript Poly-A tail Cut intron 3. The 5´ cap of the mRNA associates with the small subunit of the ribosome. The initiator tRNA and large subunit are added to form an initiation complex. Mature mRNA 5´ cap Large subunit 5´ cap mRNA Small subunit Cytoplasm Cytoplasm Amino acids tRNA arrivesin A site 3´ mRNA 5´ A site P site E site 4. The ribosome cycle begins with the growing peptide attached to the tRNA in the P site. The next charged tRNA binds to the A site with its anticodon complementary to the codon in the mRNA in this site. 64 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA RNA polymerase polymerase IIII 1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript. 3´ 5´ Primary Primary RNA RNA transcript transcript 2. The primary transcript is processed by addition of a 5´ methyl-G cap, cleavage and polyadenylation of the 3´ end, and removal of introns. The mature mRNA is then exported through nuclear pores to the cytoplasm. Primary RNA transcript Poly-A tail Cut intron 3. The 5´ cap of the mRNA associates with the small subunit of the ribosome. The initiator tRNA and large subunit are added to form an initiation complex. Cytoplasm Amino acids tRNA arrivesin A site 3´ Mature mRNA 5´ cap Large subunit 5´ cap mRNA Small subunit Cytoplasm Lengthening polypeptide chain Emptyt RNA 3´ mRNA 5´ A site P site E site 4. The ribosome cycle begins with the growing peptide attached to the tRNA in the P site. The next charged tRNA binds to the A site with its anticodon complementary to the codon in the mRNA in this site. 5´ 5. Peptide bonds form between the amino terminus of the next amino acid and the carboxyl terminus of the growing peptide. This transfers the growing peptide to the tRNA in the A site, leaving the tRNA in the P site empty. 65 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA RNA polymerase polymerase IIII 1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript. 3´ 5´ Primary Primary RNA RNA transcript transcript 2. The primary transcript is processed by addition of a 5´ methyl-G cap, cleavage and polyadenylation of the 3´ end, and removal of introns. The mature mRNA is then exported through nuclear pores to the cytoplasm. Primary RNA transcript Poly-A tail Cut intron 5´ cap 3. The 5´ cap of the mRNA associates with the small subunit of the ribosome. The initiator tRNA and large subunit are added to form an initiation complex. Cytoplasm Amino acids tRNA arrivesin A site 3´ Large subunit 5´ cap mRNA Small subunit Cytoplasm Empty tRNA moves into E site and is ejected Lengthening polypeptide chain Emptyt RNA Mature mRNA 3´ 3´ mRNA 5´ A site P site E site 4. The ribosome cycle begins with the growing peptide attached to the tRNA in the P site. The next charged tRNA binds to the A site with its anticodon complementary to the codon in the mRNA in this site. 5´ 5. Peptide bonds form between the amino terminus of the next amino acid and the carboxyl terminus of the growing peptide. This transfers the growing peptide to the tRNA in the A site, leaving the tRNA in the P site empty. 5´ 6. Ribosome translocation moves the ribosome relative to the mRNA and its bound tRNAs. This moves the growing chain into the P site, leaving the empty tRNA in the E site and the A site ready to bind the next charged tRNA. 66 67 Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the “Normal” or “Slide Sorter” views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at http://get.adobe.com/flashplayer. 68 Mutation: Altered Genes • Point mutations alter a single base • Base substitution – substitute one base for another – Silent mutation – same amino acid inserted – Missense mutation – changes amino acid inserted • Transitions • Transversions – Nonsense mutations – changed to stop codon 69 70 71 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Normal Deoxygenated Tetramer Normal HBB Sequence Polar Leu C T Thr G A C Pro T C C Glu T G A Glu G A A Lys G A A Ser G T C Amino acids T Nucleotides Abnormal Deoxygenated Tetramer 1 2 1 2 1 2 1 2 Hemoglobin tetramer "Sticky" nonpolar sites Abormal HBB Sequence Nonpolar (hydrophobic) Leu C T Thr G A C val Pro T C C T G T Glu G A A Lys G A A Ser G T C Amino acids T Nucleotides Tetramers form long chains when deoxygenated. This distorts the normal red blood cell shape into a sickle shape. 72 • Frameshift mutations – Addition or deletion of a single base – Much more profound consequences – Alter reading frame downstream – Triplet repeat expansion mutation • Huntington disease • Repeat unit is expanded in the disease allele relative to the normal 73 Chromosomal mutations • Change the structure of a chromosome – Deletions – part of chromosome is lost – Duplication – part of chromosome is copied – Inversion – part of chromosome in reverse order – Translocation – part of chromosome is moved to a new location 74 75 76 • Mutations are the starting point for evolution • Too much change, however, is harmful to the individual with a greatly altered genome • Balance must exist between amount of new variation and health of species 77