Biology Sylvia S. Mader Michael Windelspecht

Biology Sylvia S. Mader Michael Windelspecht
Chapter 12 Molecular Biology of the Gene Lecture Outline See separate FlexArt PowerPoint slides for all figures and tables pre-inserted into PowerPoint without notes. 1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1

12.1 The Genetic Material Frederick Griffith investigated virulence of Streptococcus pneumoniae Concluded that virulence could be passed from a dead strain to a nonvirulent living strain Transformation Further research by Avery et al. Discovered that DNA is the transforming substance DNA from dead cells was being incorporated into the genome of living cells

The Genetic Material Griffith’s Transformation Experiment
Mice were injected with two strains of pneumococcus: an encapsulated (S) strain and a non-encapsulated (R) strain. The S strain is virulent (the mice died); it has a mucous capsule and forms “shiny” colonies. The R strain is not virulent (the mice lived); it has no capsule and forms “dull” colonies.

Griffith’s Transformation Experiment
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. capsule Injected live R strain has no capsule and mice do not die. Injected live S strain has capsule and causes mice to die. Injected heat- killed S strain does not cause mice to die. Injected heat-killed S strain plus live R strain causes mice to die. Live S strain is withdrawn from dead mice. a. b. c. d.

The Genetic Material Transformation of organisms today:
Result is the so-called genetically modified organisms (GMOs) Invaluable tool in modern biotechnology today Commercial products that are currently much used Green fluorescent protein (GFP) can be used as a marker A jellyfish gene codes for GFP The jellyfish gene is isolated and then transferred to a bacterium, or the embryo of a plant, pig, or mouse. When this gene is transferred to another organism, the organism glows in the dark

Transformation of Organisms

The Genetic Material DNA contains: Two Nucleotides with purine bases
Adenine (A) Guanine (G) Two Nucleotides with pyrimidine bases Thymine (T) Cytosine (C)

The Genetic Material Chargaff’s Rules:
The amounts of A, T, G, and C in DNA: Are constant among members of the same species Vary from species to species In each species, there are equal amounts of: A and T G and C All this suggests that DNA uses complementary base pairing to store genetic information Each human chromosome contains, on average, about 140 million base pairs The number of possible nucleotide sequences is 4140,000,000

Nucleotide Composition of DNA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. O NH2 C CH3 adenine (A) C N thymine (T) HN C N C CH nitrogen-containing base O C CH HC C N N N O O 5 5 HO P O CH2 O HO P O CH2 O O C H H C O C 4 1 H H C 4 1 sugar = deoxyribose H C C H H C C H NH2 3 2 O 3 2 OH H OH H C guanine (G) C N cytosine (C) N CH HN C CH O C CH H2N C C N N N O O 5 phosphate HO P O CH2 O 5 HO P O CH2 O O C H H C O C C 4 1 H H 4 1 H C C H H C H C2 3 2 3 a. Purine nucleotides OH H b. Pyrimidine nucleotides OH H DNA Composition in Various Species (%) 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

The Genetic Material X-Ray diffraction:
Rosalind Franklin studied the structure of DNA using X-rays. She found that if a concentrated, viscous solution of DNA is made, it can be separated into fibers. Under the right conditions, the fibers can produce an X-ray diffraction pattern She produced X-ray diffraction photographs. This provided evidence that DNA had the following features: DNA is a helix. Some portion of the helix is repeated.

X-Ray Diffraction of DNA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Rosalind Franklin diffraction pattern diffracted X-rays a. X-ray beam Crystalline DNA c. b. © Photo Researchers, Inc.; c: © Science Source/Photo Researchers, Inc.

The Genetic Material The Watson and Crick Model (1953)
Double helix model is similar to a twisted ladder Sugar-phosphate backbones make up the sides Hydrogen-bonded bases make up the rungs Complementary base pairing ensures that a purine is always bonded to a pyrimidine (A with T, G with C) Received a Nobel Prize in 1962

Watson and 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 T 5′ end 3′ end P A G C P S S A T P P T A P S G S P C 3′ end 5′ end P complementary base pairing C c . G P hydrogen bonds sugar a: © Photodisk Red/Getty RF; d: © A. Barrington Brown/Photo Researchers

12.2 Replication of DNA DNA replication is the process of copying a DNA molecule. Semiconservative replication - each strand of the original double helix (parental molecule) serves as a template (mold or model) for a new strand in a daughter molecule.

Semiconservative Replication
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 DNA polymerase enzyme A T A T G G T C G C G C G C A A C A T C G C region of replication: new nucleotides are pairing with those of parental strands T G A A T T A T T A G C T C A T A G A T A T A A G A G G T region of completed replication T A C C C A G C 3′ G n e w o l d A s t r a n d s t r a n d 5′ daughter DNA double helix old strand new strand daughter DNA double helix

Replication of DNA Replication requires the following steps:
Unwinding, or separation of the two strands of the parental DNA molecule Complementary base pairing between a new nucleotide and a nucleotide on the template strand Joining of nucleotides to form the new strand Each daughter DNA molecule contains one old strand and one new strand 16

Replication of DNA Prokaryotic Replication
Bacteria have a single circular loop of DNA Replication moves around the circular DNA molecule in both directions Produces two identical circles The process begins at the origin of replication Replication takes about 40 minutes, but the cell divides every 20 minutes A new round of replication can begin before the previous round is completed

Replication of DNA Eukaryotic Replication
DNA replication begins at numerous points along each 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 a new strand paired with each old strand Requires enzyme DNA polymerase

Replication of DNA Eukaryotic Replication
Replication bubbles spread bidirectionally until they meet The complementary nucleotides are joined 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.

Replication of DNA Accuracy of Replication
DNA polymerase is very accurate, yet makes a mistake about once per 100,000 base pairs. Capable of identifying and correcting errors

12.3 The Genetic Code of Life
Genes Specify Enzymes Beadle and Tatum: Experiments on the fungus Neurospora crassa Proposed that each gene specifies the synthesis of one enzyme One-gene-one-enzyme hypothesis

The Genetic Code of Life
The mechanism of gene expression DNA in genes specify information, but information is not structure and function Genetic information is expressed into structure and function through protein synthesis The expression of genetic information into structure and function: DNA in a gene determines the sequence of nucleotides in an RNA molecule RNA controls the primary structure of a protein

The Central Dogma of Molecular Biology
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. nontemplate strand 5 3 A G C G A C C C C DNA T C G C T G G G G 3 5 template strand transcription in nucleus 5 3 A G C G A C C C C mRN A translation at ribosome codon 1 codon 2 codon 3 O O O polypeptide N C C N C C N C C R1 R2 R3 23 Serine Aspartate Proline

RNA is a polymer of RNA nucleotides RNA nucleotides contain the sugar ribose instead of deoxyribose RNA nucleotides are of four types: uracil (U), adenine (A), cytosine (C), and guanine(G) Uracil (U) replaces thymine (T) of DNA Types of RNA Messenger (mRNA) - Takes a message from DNA in the nucleus to ribosomes in the cytoplasm Ribosomal (rRNA) - Makes up ribosomes, which read the message in mRNA Transfer (tRNA) - Transfers the appropriate amino acid to the ribosomes for protein synthesis

Structure of RNA 5′ end G U S A base is uracil instead of thymine C S
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5′ end P G U S A base is uracil instead of thymine P C S P S P S ribose 3′ end one nucleotide

RNA Structure Compared to DNA structure

The unit of the genetic 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”

Messenger RNA Codons First Base Second Base Third Base U C A G UUU
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. First Base Second Base Third Base U C A G UUU phenylalanine UCU serine UAU tyrosine UGU cysteine U UUC phenylalanine UCC serine UAC tyrosine UGU cysteine C U 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 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 A 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 G GUA valine GCA alanine GAA glutamate GGA glycine A GUG valine GCG alanine GAG glutamate GGG glycine G

Properties of the genetic code: Universal With few exceptions, all organisms use the code the same way Encode the same 20 amino acids with the same 64 triplets Degenerate (redundant) There are 64 codons available for 20 amino acids Most amino acids encoded by two or more codons Unambiguous (codons are exclusive) 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

12.4 First Step: Transcription
A segment of DNA serves as a template for the production of an RNA molecule The gene unzips and exposes unpaired bases Serves as template for mRNA formation Loose RNA nucleotides bind to exposed DNA bases using the C=G and A=U rule When entire gene is transcribed into mRNA, the result is a pre-mRNA transcript of the gene The base sequence in the pre-mRNA is complementary to the base sequence in DNA

First Step: Transcription
A single chromosome 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 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 segment of DNA corresponding to a gene is unzipped to expose the bases of the sense strand 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

Transcription 3′ 5′ terminator gene strand template strand T C G T 3′
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3′ 5′ terminator gene strand template strand T C G T 3′ C G direction of polymerase movement A A T RNA polymerase T DNA template strand mRNA transcript promoter 5′ 5′ 3′ to RNA processing

© Oscar L. Miller/Photo Researchers, Inc.
RNA Polymerase Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. a. 200m spliceosome DNA RNA polymerase RNA transcripts © Oscar L. Miller/Photo Researchers, Inc. b.

Pre-mRNA is modified before leaving the eukaryotic nucleus. Modifications to the ends of the primary transcript: Cap on the 5′ end The cap is a modified guanine (G) nucleotide Helps a ribosome determine where to attach when translation begins Poly-A tail of adenines on the 3′ end Facilitates the transport of mRNA out of the nucleus Inhibits degradation of mRNA by hydrolytic enzymes.

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: Some segments that will not be expressed (introns) Segments that will be expressed (exons) Performed by spliceosome complexes in nucleoplasm Introns are excised Remaining exons are spliced back together Result is a mature mRNA transcript

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

Messenger RNA Processing in Eukaryotes
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. exon exon exon DNA intron intron transcription exon exon exon pre-mRNA 5 intron intron 3 exon exon exon 5 3 cap intron intron poly-A tail spliceosome exon exon exon 5 3 cap poly-A tail pre-mRNA splicing intron RNA mRNA 5 3 cap poly-A tail nuclear pore in nuclear envelope nucleus 37 cytoplasm

Functions of introns: As organismal complexity increases; The number of protein-coding genes does not keep pace But the proportion of the genome that is introns increases Possible functions of introns: Exons might combine in various combinations Would allow different mRNAs to result from one segment of DNA Introns might regulate gene expression Introns may encourage crossing-over during meiosis Exciting new picture of the genome is emerging

12.5 Second Step: Translation
The sequence of codons in the mRNA at a ribosome directs the sequence of amino acids into a polypeptide A nucleic acid sequence is translated into a protein sequence 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 types of tRNA An mRNA transcript associates with the rRNA of a ribosome in the cytoplasm or a ribosome associated with the rough endoplasmic reticulum The ribosome “reads” the information in the transcript Ribosome directs various types of tRNA to bring in their specific amino acid “fares” The tRNA specified is determined by the code being translated in the mRNA transcript

Second Step: Translation
tRNA molecules come in 64 different kinds All are very similar except that One end bears a specific triplet (of the 64 possible) called the anticodon The other end binds with a specific amino acid type tRNA synthetases attach the correct amino acid to the correct tRNA molecule All tRNA molecules with a specific anticodon will always bind with the same amino acid

Structure of a tRNA Molecule
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 G A A C A G U C C U U C C U C mRNA 5’ 3’ codon 41 a. b.

Ribosomes Ribosomal RNA (rRNA): Produced from a DNA template in the nucleolus of a nucleus 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

Ribosome Structure and Function
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. large subunit 5 3 A P E mRNA tRNA binding sites small subunit a. Structure of a ribosome b. Binding sites of ribosome outgoing tRNA polypeptide incoming tRNA mRNA c. Function of ribosomes d. Polyribosome Courtesy Alexander Rich

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

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

Initiation amino acid methionine initiator tRNA U A 5 A C mRNA E site
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. amino acid methionine Met initiator tRNA U A 5 A C mRNA E site P site A site U G 3 Met small ribosomal subunit large ribosomal subunit U A C A U G 5 start codon 3 A small ribosomal subunit binds to mRNA; an initiator tRNA pairs with the mRNA start codon AUG. The large ribosomal subunit completes the ribosome. Initiator tRNA occupies the P site. The A site is ready for the next tRNA. Initiation

Second Step: 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 The incoming tRNA joins the ribosome at its A site Methionine of the initiator tRNA is connected to the amino acid of the 2nd tRNA by a peptide bond

Second Step: Elongation
The second tRNA moves to P site (translocation) The spent initiator tRNA moves to the E site and exits The 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 its A site The dipeptide on the 2nd amino acid is connected to the amino acid of the 3rd tRNA by a peptide bond

Elongation 49 peptide bond tRNA peptide anticodon bond 1
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Met Met asp Met Met Ser Thr peptide bond tRNA Ser Ser Ser Ala Ala Ala C U G Ala Trp peptide Trp U G Trp anticodon Trp bond G Val Val Val Val Asp Asp Asp A U C A U C A U C U G C C A U C U G C U G G U A G A C G U A G A C G U A G A C G U A G A C A C C 3 5 3 5 5 3 5 3 1 A tRNA–amino acid approaches the ribosome and binds at the A site. 2 Two tRNAs can be at a ribosome at one time; the anticodons are paired to the codons. 3 Peptide bond formation attaches the peptide chain to the newly arrived amino acid. 4 The ribosome moves forward; the “empty” tRNA exits from the E site; the next amino acid–tRNA complex is approaching the ribosome. Elongation 49

Termination: Previous tRNA moves to the P site Spent tRNA moves to the 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 A protein called a release factor binds to the stop codon and cleaves the polypeptide from the last tRNA The ribosome releases the mRNA and dissociates into subunits The same mRNA may be read by another ribosome

Termination release factor stop codon Termination 5′ 3′
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Asp Ala Trp release factor Asp Val Ala Glu Trp Val U U A A Glu U G A 5′ stop codon 3′ The ribosome comes to a stop codon on the mRNA. A release factor binds to the site. U C U A A U G G A 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

Summary of Protein Synthesis in Eukaryotes
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. TRANSCRIPTION TRANSLATION 1. DNA in nucleus serves as a template for mRNA. DN A 3. mRNA moves into cytoplasm and becomes associated with ribosomes. 2. mRNA is processed before leaving the nucleus. large and small ribosomal subunits 5 mRNA introns pre-mRN A 3 mRN A amino acids 4. tRNAs with anticodons carry amino acids to mRNA. nuclear pore peptide ribosome tRNA U A C U A C 5 A U G 3 anticodon codon 5. During initiation, anticodon-codon complementary base pairing begins as the ribosomal subunits come together at a start codon. 8. During termination, a ribosome reaches a stop codon; mRNA and ribosomal subunits disband. C C C 5 C C C U G G U U U G G G A C C A A A G U A 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.

12.6 Structure of the Eukaryotic Chromosome
Each 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 The most abundant proteins in chromosomes Five primary types of histone molecules Responsible for packaging the DNA The DNA double helix is wound at intervals around a core of eight histone molecules (called a nucleosome) Nucleosomes are joined by “linker” DNA.

Structure of Eukaryotic Chromosomes
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2nm DNA double helix 11 nm 1. Wrapping of DNA around histone proteins. a. Nucleosomes (“beads on a string”) histones nucleosome histone H1 2. Formation of a three-dimensional zigzag structure via histone H1 and other DNA-binding proteins. b. 30-nm fiber 30 nm 300 nm 3. Loose coiling into radial loops . c. Radial loop domains euchromatin 4. Tight compaction of radial loops to form heterochromatin. 700 nm d. Heterochromatin 5. Metaphase chromosome forms with the help of a protein scaffold. 1,400 nm e. Metaphase chromosome a: © Ada L. Olins and Donald E. Olins/Biological Photo Service; b: Courtesy Dr. Jerome Rattner, Cell Biology and Anatomy, University of Calgary; c: Courtesy of Ulrich Laemmli and J.R. Paulson, Dept. of Molecular Biology, University of Geneva, Switzerland; d: © Peter Engelhardt/Department of Pathology and Virology, Haartman Institute/Centre of Excellence in Computational Complex Systems Research, Biomedical Engineering and Computational Science, Faculty of Information and Natural Sciences, Helsinki University of Technology, Helsinki, Finland

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