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Chapter 12: pp. 211 - 232 0.34 nm 3.4 nm 2 nm b. sugar-phosphate backbone C G T A MCAT Prep. Exam Molecular Genetics P A T P G C P C P G Sugar hydrogen bonds PowerPoint® Lecture Slides are prepared by Dr. Isaac Barjis, Biology Instructor 1 Structure of DNA DNA contains: Two Nucleotides with purine bases Adenine (A) Guanine (G) Two Nucleotides with pyrimidine bases Thymine (T) Cytosine (C) 2 Chargaff’s Rules The amounts of A, T, G, and C in DNA: Identical in identical twins Varies between individuals of a species Varies more from species to species In each species, there are equal amounts of: A&T G&C All this suggests DNA uses complementary base pairing to store genetic info Human chromosome estimated to contain, on average, 140 million base pairs Number of possible nucleotide sequences, 4,140,000,000 3 Nucleotide Composition of DNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. NH 2 adenine (A) N C CH HC N O HO P O C N O H C 3 OH C C CH N O O CH 2 O C H HN nitrogen-containing base 5 4 CH 3 C thymine (T) N C HO O H C1 C H 2 guanine (G) HN C 5 O CH 2 O 4 C H H C 3 O H P OH H C1 C H sugar = deoxyribose NH 2 2 H C cytosine (C) N C CH H2N C N O 4 C H H C 3 OH a. Purine nucleotides Species CH C N O O CH 2 O O DNA Composition in O C N 5 HO P phosphate CH N HO O H C1 C H 5 O CH 2 O 4 C H H C 3 2 H P b. Pyrimidine nucleotides H C1 C H 2 OH H Various Species (%) A T G C Homo sapiens (human) 31.0 31.5 19.1 18.4 Drosophila melanogaster (fruit fly) 27.3 27.6 22.5 22.5 Zea mays (corn) 25.6 25.3 24.5 24.6 Neurospora crassa (fungus) 23.0 23.3 27.1 26.6 Escherichia coli (bacterium) 24.6 24.3 25.5 25.6 Bacillus subtilis (bacterium) 28.4 29.0 21.0 21.6 c. Chargaff’s data 4 Watson and Crick Model Watson and Crick, 1953 Constructed a model of DNA Double-helix model is similar to a twisted ladder Sugar-phosphate backbones make up the sides Hydrogen-bonded bases make up the rungs Received a Nobel Prize in 1962 5 Watson/Crick Model of DNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3.4 nm 0.34 nm 2 nm b. d. C a. sugar-phosphate backbone G 5 4 2 1 S 3 2 T 3 end 5 end P G 3 C 1 P A 4 S A 5 T P P c. G C P C complementary base G P hydrogen bonds sugar a: © Kenneth Eward/Photo Researchers, Inc.; d: © A. Barrington Brown/Photo Researchers, Inc. 6 Replication of DNA DNA replication is the process of copying a DNA molecule. Replication is semiconservative, with each strand of the original double helix (parental molecule) serving as a template (mold or model) for a new strand in a daughter molecule. 7 Replication: Eukaryotic DNA replication begins at numerous points along linear chromosome DNA unwinds and unzips into two strands Each old strand of DNA serves as a template for a new strand Complementary base-pairing forms new strand on each old strand Requires enzyme DNA polymerase 8 Replication: Eukaryotic Replication bubbles spread bi-directionally until they meet The complementary nucleotides join to form new strands. Each daughter DNA molecule contains an old strand and a new strand. Replication is semiconservative: One original strand is conserved in each daughter molecule i.e. each daughter double helix has one parental strand and one new strand. 9 Semiconservative Replication of DNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5' 3' G C G C G A T A region of parental DNA double helix T A C G A T A G C C G G A region of replication: new nucleotides are pairing with those of parental strands C G C A A G T T A T T G A C A A T T C A T A C G C G A G C G A old strand A G region of completed replication A T G T A T A C new strand new strand old strand daughter DNA double helix daughter DNA double helix 10 Science Focus: Aspects of DNA Replication Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. P is attached here base is attached here OH 5 CH2 4 C H 3 O OH C1 H H C 1 H C 2 H OH Deoxyribose molecule 2 5 end P DNA polymerase attaches a new nucleotide to the 3 carbon of the previous nucleotide. P P G P C 3 end P C P G 5 3 P P T A P C G 3 end P 5 end template strand Direction of replication P template strand 4 DNA polymerase leading new strand 3 new strand 3 helicase at replication fork RNA primer template strand 5 6 Okazaki fragment 3 lagging strand 5 5 7 DNA ligase 3 Replication fork introduces complications parental DN A helix DNA polymerase 11 Replication: Prokaryotic Prokaryotic Replication Bacteria have a single circular loop Replication moves around the circular DNA molecule in both directions Produces two identical circles Cell divides between circles, as fast as every 20 minutes 12 Replication: Prokaryotic vs. Eukaryotic Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. origin replication is complete replication is occurring in two directions a. Replication in prokaryotes replication fork replication bubble parental strand new DNA duplexes b. Replication in eukaryotes daughter strand 13 Replication Errors Genetic variations are the raw material for evolutionary change Mutation: A permanent (but unplanned) change in basepair sequence Some due to errors in DNA replication Others are due to to DNA damage DNA repair enzymes are usually available to reverse most errors 14 Function of Genes Genes Specify Enzymes Beadle and Tatum: Experiments on fungus Neurospora crassa Proposed that each gene specifies the synthesis of one enzyme One-gene-one-enzyme hypothesis Genes Specify a Polypeptide A gene is a segment of DNA that specifies the sequence of amino acids in a polypeptide Suggests that genetic mutations cause changes in the primary structure of a protein 15 Protein Synthesis: From DNA to RNA to Protein The mechanism of gene expression DNA in genes specify information, but information is not structure and function Genetic info is expressed into structure & function through protein synthesis The expression of genetic info into structure & function: DNA in gene controls the sequence of nucleotides in an RNA molecule RNA controls the primary structure of a protein 16 The Central Dogma of Molecular Biology Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. nontemplate strand 5' 3' A G G G A C C C C T C G C T G G G G DNA 3' 5' transcription in nucleus template strand 5' mRN 3' A G G codon 1 translation at ribosome A C C C codon 2 O N polypeptide G C C codon 3 O N C C O N C R1 R2 R3 Serine Aspartate Proline C 17 Types of RNA RNA is a polymer of RNA nucleotides RNA Nucleotides are of four types: Uracil, Adenine, Cytosine, and Guanine Uracil (U) replaces thymine (T) of DNA Types of RNA Messenger (mRNA) - Takes genetic message from DNA in nucleus to ribosomes in cytoplasm Ribosomal (rRNA) - Makes up ribosomes which read the message in mRNA Transfer (tRNA) - Transfers appropriate amino acid to ribosome when “instructed” 18 Structure of RNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. G P G S U A P U base is uracil instead of thymine C S P A S P C S ribose one nucleotide 19 RNA vs. DNA structure 20 The Genetic Code Properties of the genetic code: Universal Degenerate (redundant) There are 64 codons available for 20 amino acids Most amino acids encoded by two or more codons Unambiguous (codons are exclusive) With few exceptions, all organisms use the code the same way Encode the same 20 amino acids with the same 64 triplets None of the codons code for two or more amino acids Each codon specifies only one of the 20 amino acids Contains start and stop signals Punctuation codons Like the capital letter we use to signify the beginning of a sentence, and the period to signify the end 21 The Genetic Code The unit of a code consists of codons, each of which is a unique arrangement of symbols Each of the 20 amino acids found in proteins is uniquely specified by one or more codons The symbols used by the genetic code are the mRNA bases Function as “letters” of the genetic alphabet Genetic alphabet has only four “letters” (U, A, C, G) Codons in the genetic code are all three bases (symbols) long Function as “words” of genetic information Permutations: There are 64 possible arrangements of four symbols taken three at a time Often referred to as triplets Genetic language only has 64 “words” 22 Messenger RNA Codons Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. First Base U C A G Second Base A G Third Base U C UUU phenylalanine UCU serine UAU tyrosine UGU cysteine U UUC phenylalanine UCC serine UAC tyrosine UGU cysteine C UUA leucine UCA serine UAA stop UGA stop A UUG leucine UCG serine UAG stop UGG tryptophan G CUU leucine CCU proline CAU histidine CGU arginine U CUC leucine CCC proline CAC histidine CGC arginine C CUA leucine CCA proline CAA glutamine CGA arginine A CUG leucine CCG proline CAG glutamine CGG arginine G AUU isoleucine ACU threonine AAU asparagine AGU serine U AUC isoleucine ACC threonine AAC asparagine AGC serine C AUA isoleucine ACA threonine AAA lysine AGA arginine A AUG (start) methionine ACG threonine AAG lysine AGG arginine G GUU valine GCU alanine GAU aspartate GGU glycine U GUC valine GCC alanine GAC aspartate GGC glycine C GUA valine GCA alanine GAA glutamate GGA glycine A GUG valine GCG alanine GAG glutamate GGG glycine G 23 Steps in Gene Expression: Transcription Transcription Gene unzips and exposes unpaired bases Serves as template for mRNA formation Loose RNA nucleotides bind to exposed DNA bases using the C=G & A=U rule When entire gene is transcribed into mRNA, result is a pre-mRNA transcript of the gene The base sequence in the pre-mRNA is complementary to the base sequence in DNA 24 Transcription of mRNA A single chromosomes consists of one very long molecule encoding hundreds or thousands of genes The genetic information in a gene describes the amino acid sequence of a protein The segment of DNA corresponding to a gene is unzipped to expose the bases of the sense strand The information is in the base sequence of one side (the “sense” strand) of the DNA molecule The gene is the functional equivalent of a “sentence” The genetic information in the gene is transcribed (rewritten) into an mRNA molecule The exposed bases in the DNA determine the sequence in which the RNA bases will be connected together RNA polymerase connects the loose RNA nucleotides together The completed transcript contains the information from the gene, but in a mirror image, or complementary form 25 Transcription Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5' C C G A T A T nontemplate strand template strand 3' A C G C G G A G RNA polymerase DNA template strand mRNA transcript C A T C T G A 5' 3' to RNA processing 26 Processing Messenger RNA Pre-mRNA, is modified before leaving the eukaryotic nucleus. Modifications to ends of primary transcript: Cap of modified guanine on 5′ end The cap is a modified guanine (G) nucleotide Helps a ribosome where to attach when translation begins Poly-A tail of 150+ adenines on 3′ end Facilitates the transport of mRNA out of the nucleus Inhibits degradation of mRNA by hydrolytic enzymes. 27 Processing Messenger RNA Pre-mRNA, is composed of exons and introns. The exons will be expressed, The introns, occur in between the exons. Allows a cell to pick and choose which exons will go into a particular mRNA RNA splicing: Primary transcript consists of: Performed by spliceosome complexes in nucleoplasm Some segments that will not be expressed (introns) Segments that will be expressed (exons) Introns are excised Remaining exons are spliced back together Result is mature mRNA transcript 28 RNA Splicing In prokaryotes, introns are removed by “self-splicing”—that is, the intron itself has the capability of enzymatically splicing itself out of a pre-mRNA 29 Messenger RNA Processing Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. exon exon exon DNA intron intron transcription exon pre-mRNA intron intron 5' exon exon exon 3' exon exon 3' 5' cap poly-A tail intron intron spliceosome exon exon exon 3' 5' cap poly-A tail pre-mRNA splicing intron RNA mRNA 5' 3' cap poly-A tail nuclear pore in nuclear envelope nucleus cytoplasm 30 Functions of Introns As organismal complexity increases; Number of protein-coding genes does not keep pace But the proportion of the genome that is introns increases Humans: Possible functions of introns: More bang for buck Genome has only about 25,000 coding genes Up to 95% of this DNA genes is introns Exons might combine in various combinations Would allow different mRNAs to result from one segment of DNA Introns might regulate gene expression Exciting new picture of the genome is emerging 31 Steps in Gene Expression: Translation tRNA molecules have two binding sites One associates with the mRNA transcript The other associates with a specific amino acid Each of the 20 amino acids in proteins associates with one or more of 64 species of tRNA Translation An mRNA transcript migrates to rough endoplasmic reticulum Associates with the rRNA of a ribosome The ribosome “reads” the information in the transcript Ribosome directs various species of tRNA to bring in their specific amino acid “fares” tRNA specified is determined by the code being translated in the mRNA transcript 32 tRNA tRNA molecules come in 64 different kinds All very similar except that One end bears a specific triplet (of the 64 possible) called the anticodon Other end binds with a specific amino acid type tRNA synthetases attach correct amino acid to the correct tRNA molecule All tRNA molecules with a specific anticodon will always bind with the same amino acid 33 Structure of tRNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. amino acid leucine 3 5 Hydrogen bonding amino acid end anticodon anticodon end mRNA 5' 3' codon b. 34 Ribosomes Ribosomal RNA (rRNA): Produced from a DNA template in the nucleolus Combined with proteins into large and small ribosomal subunits A completed ribosome has three binding sites to facilitate pairing between tRNA and mRNA The E (for exit) site The P (for peptide) site, and The A (for amino acid) site 35 Ribosomal Structure and Function Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. large subunit 3 5 mRNA tRNA binding sites small subunit outgoing tRNA b. Binding sites of ribosome a. Structure of a ribosome polypeptide incoming tRNA incoming tRNA c. Function of ribosomes d. Polyribosome Courtesy Alexander Rich 36 Steps in Translation: Initiation Components necessary for initiation are: Small ribosomal subunit mRNA transcript Initiator tRNA, and Large ribosomal subunit Initiation factors (special proteins that bring the above together) Initiator tRNA: Always has the UAC anticodon Always carries the amino acid methionine Capable of binding to the P site 37 Steps in Translation: Initiation Small ribosomal subunit attaches to mRNA transcript Beginning of transcript always has the START codon (AUG) Initiator tRNA (UAC) attaches to P site Large ribosomal subunit joins the small subunit 38 Steps in Translation: Initiation Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. amino acid methionine Met initiator tRNA E site P site A site mRNA 5' Met 3' small ribosomal subunit large ribosomal subunit U A C A UG 5' A small ribosomal subunit binds to mRNA; an initiator tRNA pairs with the mRNA start codon AUG. start codon 3' The large ribosomal subunit completes the ribosome. Initiator tRNA occupies the P site. The A site is ready for the next tRNA. Initiation 39 Steps in Translation: Elongation “Elongation” refers to the growth in length of the polypeptide RNA molecules bring their amino acid fares to the ribosome Ribosome reads a codon in the mRNA Allows only one type of tRNA to bring its amino acid Must have the anticodon complementary to the mRNA codon being read Joins the ribosome at it’s A site Methionine of initiator is connected to amino acid of 2nd tRNA by peptide bond 40 Steps in Translation: Elongation Second tRNA moves to P site (translocation) Spent initiator moves to E site and exits Ribosome reads the next codon in the mRNA Allows only one type of tRNA to bring its amino acid Must have the anticodon complementary to the mRNA codon being read Joins the ribosome at it’s A site Dipeptide on 2nd amino acid is connected to amino acid of 3nd tRNA by peptide bond 41 Steps in Translation: Elongation Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Met asp Met Met peptide Ser bond Ser C U G Trp A tRNA–amino acid approaches the ribosome and binds at the A site. C A U G U A 3 6 2 Two tRNAs can be at a ribosome at one time; the anticodons are paired to the codons. Val Asp Asp C A U C U G G U A G A C G A C 3 1 Asp Trp peptide bond Val Val Val C A U G U A Trp Trp anticodon Ala Ala Ala C U G C U G G A C G U A 3 6 3 Thr Ser Ser tRNA Ala Met Peptide bond formation attaches the peptide chain to the newly arrived amino acid. 4 G A C A C C 6 3 The ribosome moves forward; the “empty” tRNA exits from the E site; the next amino acid–tRNA complex is approaching the ribosome. Elongation 42 Steps in Translation: Termination Previous tRNA moves to P site Spent tRNA moves to E site and exits Ribosome reads the STOP codon at the end of the mRNA UAA, UAG, or UGA Does not code for an amino acid Polypeptide is released from last tRNA by release factor Ribosome releases mRNA and dissociates into subunits mRNA read by another ribosome 43 Steps in Translation: Termination Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Asp Ala release factor Trp Val Glu U U AA UGA stop codon 5' 3' The ribosome comes to a stop codon on the mRNA. A release factor binds to the site. 3 5 The release factor hydrolyzes the bond between the last tRNA at the P site and the polypeptide, releasing them. The ribosomal subunits dissociate. Termination 44 Summary of Gene Expression (Eukaryotes) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. TRANSCRIPTION 1. DNA in nucleus serves as a template for mRNA. TRANSLATION DNA 2. mRNA is processed before leaving the nucleus. mRNA large and small ribosomal subunits 3. mRNA moves into cytoplasm and becomes associated with ribosomes. 5 introns pre-mRNA 3' mRNA amino acids peptide nuclear pore ribosome tRNA 5 UA C A UG 4. tRNAs with anticodons carry amino acids to mRNA. 3 anticodon codon 5. During initiation, anticodon-codon complementary base pairing begins as the ribosomal subunits come together at a start codon. 5 C C C U GG U U U G GG A C C A A A G 8. During termination, a ribosome reaches a stop codon; mRNA and ribosomal subunits disband. 3' 6. During elongation, polypeptide synthesis takes place one amino acid at a time. 7. Ribosome attaches to rough ER. Polypeptide enters lumen, where it folds and is modified. 45 Structure of Eukaryotic Chromosome Contains a single linear DNA molecule, but is composed of more than 50% protein. Some of these proteins are concerned with DNA and RNA synthesis, Histones, play primarily a structural role Five primary types of histone molecules Responsible for packaging the DNA DNA double helix is wound at intervals around a core of eight histone molecules (called nucleosome) Nucleosomes are joined by “linker” DNA. 46 Structure of Eukaryotic Chromosome Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2 nm DNA double helix 1 nm a. Nucleosomes (“beads on a string”) 1. Wrapping of DNA around histone proteins. histones nucleosome 2. Formation of a three-dimensional zigzag structure via histone H1 and other DNA-binding proteins. histone H1 b. 30-nm fiber 30 nm 300 nm 3. Loose coiling into radial loops. c. Radial loop domains euchromatin 700 nm 4. Tight compaction of radial loops to form heterochromatin. d. Heterochromatin 5. Metaphase chromosome forms with the help of a protein scaffold. 1,400 nm e. Metaphase chromosome 47 Review Genetic Material DNA Structure Transformation Watson and Crick DNA Replication Prokaryotic versus Eukaryotic Replication Errors Transcription Translation Structure of Eukaryotic Chromosome 48 Chapter 12: pp. 211 - 232 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 0.34 nm 3.4 nm 2 nm b. sugar-phosphate backbone C G T A MCAT Prep. Exam Molecular Genetics P A T P G C P C P G Sugar hydrogen bonds PowerPoint® Lecture Slides are prepared by Dr. Isaac Barjis, Biology Instructor 49