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Chapter 17
Section 17.1: DNA Section 17.2: RNA Section 17.3: VirusesBiochemistry in the LabBiochemistry in Perspective
Nucleic Acids
Overview
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Humans have used artificial selection to produce certain physical traits in domesticated animals and plants for thousands of years
It wasn’t until the 19th century that selection was studied scientifically
Early in the 20th century, scientists recognized that physical traits are inherited as discrete units (genes)
Figure 17.1 The First Complete Structural Model of DNA
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Deoxyribonucleic acid (DNA) was eventually recognized as the genetic information carrier
DNA structure was elucidated in 1953 by James Watson and Francis Crick Molecular biology
emerged as a new science
Figure 17.1 The First Complete Structural Model of DNA
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
• DNA carries most of the genetic instructions used in the development, functioning and reproduction of all known living organisms and many viruses.
• DNA is composed of two polydeoxynucleotide strands forming a double helix
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
A gene is part of a DNA sequence that contains the code for a gene product, protein, or RNA
The complete DNA base sequence of an organism is its genome
DNA is synthesized by replication, which involves complementary base pairing between the parental and newly synthesized strand
Figure 17.2 Two Models of DNA Structure
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
DNA RNA phenotypeprotein
Central Dogma of Molecular Biology
CELL ORGANISM
Transcription Translationin ribosome
The central dogma is generally how the flow of information works in all organisms
2. Decoding genetic information begins with synthesis of RNA (transcription)
Transcription and involves complementary base pairing of ribonucleotides to DNA bases
Each new RNA is a transcript
The total RNA transcripts for an organism comprise its transcriptome
Figure 17.3a An Overview of Genetic Information Flow
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
3. Several RNA molecules participate directly in the synthesis of protein, or translation
Messenger RNA (mRNA) specifies the primary protein sequence
Transfer RNA (tRNA) delivers the specific amino acid
Ribosomal RNA (rRNA) participates in formation of peptide bonds between amino acids
Figure 17.3b An Overview of Genetic Information Flow
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
The proteome is the entire set of proteins synthesized
4. Gene expression is the process by which cells control the timing of gene product synthesis in response to environmental or developmental cues
Figure 17.3b An Overview of Genetic Information Flow
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
DNA consists of two polydeoxynucleotide strands that wind around each other to form a right-handed double helix Each DNA nucleotide
monomer is composed of a nitrogenous base, a deoxyribose sugar, and phosphate
Figure 17.4 DNA Strand Structure
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Nucleotides are linked by 3′,5′-phosphodiester bonds
These join the 3′-hydroxyl group of one nucleotide to the 5′-phosphate of another
Figure 17.4 DNA Strand Structure
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
The antiparallel nature of the two strands allows hydrogen bonds to form between the nitrogenous bases
Two types of base pair (bp) in DNA: (1) adenine (purine) pairs with thymine (pyrimidine) and (2) the purine guanine pairs with the pyrimidine cytosine
Figure 17.5 DNA Structure
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
The dimensions of crystalline B-DNA have been precisely measured:
1. One turn of the double helix spans 3.32 nm and consists of 10.3 base pairs
Figure 17.6 DNA Structure: GC Base Pair Dimensions
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
2. Diameter of the double helix is 2.37 nm, only suitable for base pairing a purine with a pyrimidine3. The distance between adjacent base pairs is 0.29-0.30 nm
Figure 17.6 DNA Structure: AT Base Pair Dimensions
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
DNA is a relatively stable molecule with several noncovalent interactions adding to its stability
1. Hydrophobic interactions—internal base clustering2. Hydrogen bonds—formation of preferred bonds: three between CG base pairs and two between AT base pairs3. Base stacking—bases are nearly planar and stacked, allowing for weak van der Waals forces between the rings4. Hydration—water interacts with the structure of DNA to stabilize structure5. Electrostatic interactions—destabilization by negatively charged phosphates of sugar-phosphate backbone are minimized by the shielding effect of water and Mg2+
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
DNA Structure: The Nature of Mutation DNA is eminently suited for information storage
but it is not a static molecule, and it is vulnerable to certain disruptive forces that can cause mutations
Most mutations are either deleterious or neutral, but sometimes a positive mutation can occur which enhance the adaptation of the organism
https://www.youtube.com/watch?v=g02RnGXCXrQ
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Mutation types—The most common are small single base changes, also called point mutations
These are either transition or transversion mutations
Transition mutations, caused by deamination, lead to purine for purine or pyrimidine for pyrimidine substitutions
Transversion mutations, caused by alkylating agents or ionizing radiation, involve substitution of a purine for a pyrimidine or vice versa
https://www.youtube.com/watch?v=xYOK-yzUWSI
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Point mutations that occur in a population with any frequency are referred to as single nucleotide polymorphisms (SNPs)
Point mutations that occur within the coding portion of a gene can be classified according to their impact on structure and/or function:
Silent mutations have no discernable effect Missense mutations have an observable
effect (e.g., coding for a different amino acid, changing the function of the protein)
Nonsense mutations changes a codon for an amino acid to that of a premature stop codon (protein will be incomplete)
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Insertions and deletions, or indels, occur from one to thousands of bases
Indels that occur within the coding region that are not divisible by three cause a frameshift mutation
Genome rearrangements can cause disruptions in gene structure or regulation.
Occur as a result of double strand breaks and can lead to inversions, translocations, or duplications
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Inversions result when deleted DNA is reinserted into its original position in the opposite orientation
Translocation is when a DNA fragment inserts else where in the genome
Duplication is the creation of duplicate genes or parts of genes.
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Causes of DNA Damage—DNA damage can result from exogenous and endogenous forces
Endogenous sources can include tautomeric shifts, depurination, deamination, and ROS-induced oxidative damage
Exogenous factors such as radiation and xenobiotic exposure can also be mutagenic
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Tautomeric shifts are spontaneous changes to nucleotide base structure
Amino to imino groups and keto to enol groups If tautomers form during replication, base
mispairings can occur The imino form of adenine does not pair with
thymine; it pairs with cytosine Several spontaneous hydrolytic reactions can
cause DNA damage Depurination or deamination is also possible,
which can cause a mutation in the next round of replication
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Figure 17.7 A Tautomeric Shift Causes a Transition Mutation
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Ionizing radiation (e.g., UV and X-rays) can alter DNA structure
Radiation-induced damage via free radical mechanisms can cause strand breaks, DNA-protein cross linking, ring openings, and base modifications
The most common UV-induced products are thymine-thymine dimers
Figure 17.8 Thymine Dimer Structure
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Xenobiotics can damage DNA; classes include: Base analogues have structures so similar to
bases they can be incorporated into DNA Alkylating agents cause alkylation, which is
the electrophilic attack on molecules with unpaired electrons
Often add carbon-containing alkyl groups Often results in incorrect base pairing,
leading to transition or transversion mutations
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Nonalkylating agents—a variety of other chemicals can modify DNA structure
For example, nitrous acid and sodium nitrite can deaminate bases
Intercalating agents are planar polycyclic aromatic molecules that can distort DNA by inserting themselves between the stacked bases
Causes base pair deletion or insertion The intercalating agent ethidium bromide is a
fluorescent tag molecule used as a nucleic acid stain
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
DNA Structure: The Genetic Material
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Determining the structure of DNA became an obvious priority
Investigators including Linus Pauling, Maurice Wilkins, Rosalind Franklin, James Watson, and Francis Crick all worked toward this goal
Watson and Crick won the race for the double helix and published their findings in the journal Nature in 1953
They and Wilkins were awarded the Nobel Prize in chemistry in 1962
Figure 17.1 The First Complete Structural Model of DNA
Information used to construct the model of DNA:
1. Chemical and physical dimensions of deoxyribose, nitrogenous bases, and phosphate2. 1:1 Ratios of adenine to thymine and cytosine to guanine (Chargaff’s rules)3. X-ray diffraction studies of Rosalind Franklin
Figure 17.9 X-Ray Diffraction Study of DNA by Rosalind Franklin and R. Gosling
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
4. Wilkins and Stokes diameter and pitch estimates from X-ray diffraction5. Linus Pauling’s demonstration that proteins could exist in a helical conformation
Figure 17.9 X-Ray Diffraction Study of DNA by Rosalind Franklin and R. Gosling
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
DNA Structure: Variations on a Theme Watson and Crick’s discovery
is referred to as B-DNA (sodium salt)
Another form is the A-DNA, which forms when RNA/DNA duplexes form
Z-DNA (zigzag conformation) is left-handed DNA that can form as a result of torsion during transcription
Figure 17.10 A-DNA, B-DNA, and Z-DNA
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
DNA can form other structures, including cruciforms, which are cross-like structures, probably a result of palindromes (inverted repeats)
Packaging large DNA molecules to fit inside a cell or nucleus requires a process termed supercoiling
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
DNA Supercoiling Necessary to fit large DNA moleculues into cells Facilitates several biological processes:
packaging of DNA, replication, and transcription Linear and circular DNA can be in a relaxed or
supercoiled shape
Figure 17.11 Linear and Circular DNA and DNA Winding
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
When DNA is underwound (RH helix twisted in LH direction), it twists to the right to relieve strain, causing negative supercoiling
Most naturally occurring DNA is negatively supercoiled forming either (a) toroidal or (b) plectonemic supercoil
Winds around itself to form an interwound supercoil and has stored energy in the form of torque
Figure 17.12 Supercoils
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
This stored energy facilitates strand separation in replication and transcription
Supercoiling that forms during strand separation can be relieved by a class of enzymes called topoisomerases
Make reversible cuts that allow the supercoiled segments to unwind
Figure 17.13 Effect of Strain on a Circular DNA Molecule
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Chromosomes and Chromatin DNA is packaged into
chromosomes Prokaryotic and eukaryotic
chromosomes differ significantly
In Prokaryotes, like E. coli, chromosome is a circular DNA molecule that is supercoiled and complexed with a protein corehttps://www.youtube.com/wa
tch?v=s9HPNwXd9fk
Figure 17.15 The E. coli Chromosome Removed from a Cell
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
In the nucleoid, the chromosome is attached to the protein core in at least 40 places
This structural feature limits the unraveling of supercoiled DNA
Figure 17.15 The E. coli Chromosome Removed from a Cell
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Eukaryotes have extraordinarily large genomes when compared to prokaryotes
Chromosome number and length can vary by species
Each eukaryotic chromosome consists of a single, linear DNA molecule complexed with histone proteins to form nucleohistone
Chromatin is the term used to describe this complex
Figure 17.16 Electron Micrograph of Chromatin
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Nucleosomes are formed by the binding of DNA and histone proteins
Nucleosomes have a beaded appearance when viewed by electron micrograph
Histone proteins consist of five major classes: H1, H2A, H2B, H3, and H4
A nucleosome is positively coiled DNA wrapped around a histone core (two copies each of H2A, H2B, H3, and H4)
Figure 17.16 Electron Micrograph of Chromatin
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Each of the highly conserved histone core proteins contains a common structural feature called the histone fold
Three a-helices separated by two short unstructured elements
N-terminal tails of histones protrude from nucleosomes, can be covalently modified (methylation and acetylation)
These modifications can alter the accessibility of the DNA
Figure 17.17a The Core Histones
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
The histone core forms when two sets of H2A and H2B and H3 and H4 each form two sets of head to tail heterodimers
The H3•H4 heterodimers associate and bind DNA
Figure 17.17b The Core Histones
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
The H2A•H2B heterodimers associate with the H3•H4 tetramer, completing nucleosome assembly
Histone H1 binds to the nucleosome where the DNA enters and exits and acts as a clamp that prevents unraveling
Approximately 145 bp are in contact with the histone octamer (1.8 helical turns)
Connection between nucleosomes requires approximately 60 bp of linker DNA (may vary between 20 and 70 depending on species and tissue)
Figure 17.18 Histone H1
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
In anticipation of cell division, chromatin must be compacted into chromosomes
Nucleosomes are coiled into the 30 nm fiber, which is further coiled to form 200 nm filaments
Figure 17.19 Chromatin
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
200 nm fibers have numerous supercoiled loops attached to a central nuclear scaffold
During interphase (routine activity), chromatin can exist in one of two forms: heterochromatin (highly condensed, can be activated) or euchromatin (less condensed, some transcriptional activity)
Figure 17.20 Chromatin
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Organelle DNA—Mitochondria (most Eukaryotes) and chloroplasts (plants and algae) are semiautonomous organelles that possess DNA and their own protein-synthesizing machinery
These organelles, both of which can reproduce via binary fission, require proteins expressed by their chromosomes as well as nuclear DNA
Mitochondria and chloroplasts are believed to be descendents of free-living prokaryotes, and are susceptible to antibiotics.
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Genome Structure Size varies over an enormous range from 106 bp
(Mycoplasma) to 1010 bp (certain plants) Most prokaryotic genomes are smaller than
eukaryotic genomes Larger information-coding capacity of eukaryotic
DNA
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Genomes of organisms can vary widely in complexity and gene density
Majority of Eukaryotic genome does not code for gene products: can have introns, pseudogenes, and genome-wide repeats
Figure 17.21 Comparison of 50 kb Segments of the Genomes of Selected Eukaryotes with the Prokaryotic E. coli Genome
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Prokaryotic Genomes—Investigation of E. coli has revealed the following prokaryotic features:
1. Genome size—usually considerably less DNA and fewer genes (E. coli 4.6 megabases) than eukaryotic genomes2. Coding capacity—compact and continuous genes3. Gene expression—genes organized into operons
• Operon: Set of linked genes, regulated as a unit
Prokaryotes often contain plasmids, which are usually small, circular DNA with additional genes (e.g., antibiotic resistance)
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Organization of Eukaryotic Genome is very complex
Unique eukaryotic genome features:1. Genome size—eukaryotic genome generally larger, but size does not necessarily indicate complexity2. Coding capacity—enormous protein coding capacity; 80% of human DNA sequences have biological functions3. Coding continuity—genes include noncoding introns (between exons, the expressed sequences), which can be removed by splicing from the primary RNA transcript to produce functional RNA molecules.
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Existence of introns and exons allows eukaryotes to produce more than one polypeptide from each protein-coding gene
Alternative splicing allows for various combinations of exons to be joined to form different mRNAs
Of the 3,200 Mb of the human genome, only 38% comprise genes and related sequence
Only 4% codes for gene products Humans have about 23,000 protein coding
genes and several ncRNA genes
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
25% of known protein-coding genes are related to DNA synthesis and repair
21% signal transduction 17% general biochemical functions 38% other activities
About 80-90% of the human genome is intergenic or noncoding sequences
Figure 17.22 Human Protein-Coding Genes
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Major types of DNA regulatory sequences: Promoters are in close proximity to a start
site of a specific gene. Promote transcription. An Enhancer interacts with and stimulates
the activity of an RNA polymerase complex A Silencer inhibits transcription of its gene An Insulator blocks DNA interactions with
Enhancers and Promoters Pseudogenes are nonfunctional DNA
sequences homologous to a known protein or RNA gene
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Repetitive DNA refers to copies of DNA patterns in genome.
Two classes: tandem repeats and interspersed genome-wide repeats
Tandem repeats (satellite DNA) are DNA sequences in which multiple copies are arranged next to each other
Certain tandem repeats play structural roles like centromeres and telomeres
Some are small, like microsatellites (1-4 bp) and minisatellites (10-100 bp)
Used as markers in genetic disease, forensic investigations, and kinship
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Interspersed genome-wide repeats are repetitive sequences scattered around the genome
Often involve mobile genetic elements that can duplicate and move around the genome
Transposons and retrotransposones Transposons: DNA elements that excise
themselves and move to another site Retrotransposons: Transposons that use
an RNA transcript intermediate. Two Types: LTR (long terminal repeats): Believed to
be decayed viruses (Human Endogenous Retroviruses)
Non-LTR: Do not contain long repeats
Section 17.1: DNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
RNA is a versatile molecule, involved in protein synthesis, and plays structural and enzymatic roles as well
Differences between DNA and RNA primary structure: 1. Ribose sugar instead of
deoxyribose 2. Uracil nucleotide instead of
thymineFigure 17.23 Secondary Structure of RNA
Section 17.2: RNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
3. RNA exists as a single strand that can form complex three- dimensional structures by base pairing with itself
4. Some RNA molecules have catalytic properties, or ribozymes (e.g., self-cleavages or cleave other RNA)
https://www.youtube.com/watch?v=uMKhcBCJwlc
Figure 17.23 Secondary Structure of RNA
Section 17.2: RNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Transfer RNA Transfer RNA (tRNA) molecules
transport amino acids to ribosomes for assembly (15% of cellular RNA)
Average length: 75 bases At least one tRNA for each
amino acid The 3′ terminus and the
anticodon loop of tRNA allow the tRNA to interact with a specific AA, which lets the tRNA correctly align the AA during protein synthesis.
Figure 17.24a Transfer RNA
Section 17.2: RNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Ribosomal RNA Ribosomal RNA (rRNA) is the most abundant
RNA in living cells Eeukaryotic and prokaryotic ribosomes are
similar in shape and function, both have a small and large subunit, but differ in size and chemical composition
Eukaryotic are larger (80S) with a 60S and 40S subunit, while prokaryotic are smaller (70S) with 50S and 30S subunits (S represents centrifuge sedimentation values, which depend on both mass and shape of the molecules).
Section 17.2: RNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Messenger RNA Messenger RNA (mRNA) is the carrier of
genetic information from DNA to protein synthesis (approximately 5% of total RNA)
mRNA varies considerably in size Prokaryotic and eukaryotic mRNA differ in
several respects Prokaryotes are polycistronic (mRNA codes for
multiple polypeptides) while eukaryotes are usually monocistronic
mRNAs are processed differently; eukaryotic mRNA are extensively modified, while prokaryotic mRNAs are translated into proteins immediately.
Section 17.2: RNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Noncoding RNAs RNAs that do not directly code for polypeptides
are called noncoding RNAs (ncRNAs) Micro RNAs (miRNAs) are involved regulation of
gene expression. Small interfering RNAs (siRNAs) are involved
in the RNA-induced silencing complex that interfere RNA-containing viruses
Small Nucleolar RNAs (snoRNAs) facilitate chemical modifications to rRNA in the nucleolus
Section 17.2: RNA
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
VirusesViruses lack the properties that distinguish life from nonlife (e.g., no metabolism)
Once a virus has infected a cell, its nucleic acid can hijack the host’s nucleic acid and protein-synthesizing machinery The virus can then make copies of itself until it
ruptures the host cell or integrates into the host cell’s chromosome
https://www.youtube.com/watch?v=0h5Jd7sgQWY
Section 17.3: Viruses
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
The Structure of Viruses Simple virions are composed of a capsid, which
encloses nucleic acid Most capsids are helical or icosahedral (a
polyhedron with 20 faces) The nucleic acid is DNA or RNA
Can be single- or double-stranded, and the single-stranded RNA viruses can be positive-sense (+) or negative-sense – (e.g. (-)-ssRNA)
https://www.youtube.com/watch?v=s8jhJXgC-bk
Section 17.3: Viruses
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
(-)-ssRNA viruses need reverse transcriptase to synthesize mRNA
More complex viruses may have a membrane envelope or have proteins that protrude from the surface, called spikes
Section 17.3: Viruses
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Bacteriophage T4 is a large, icosahedral virus with a complex tail structure
Life cycle includes the lysogenic and lytic cycle Lytic cycle involves destroying the host by making copies of
itself The lysogenic cycle involves integrating the viral DNA into
the host chromosome forming a prophage
Figure 17.27 The T4 Bacteriophage
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Section 17.3: Viruses
HIV is the causative agent of acquired immunodeficiency syndrome (AIDS)
Belongs to a unique group of RNA viruses called retroviruses, which contain reverse transcriptase
Figure 17J HIV Structure
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Biochemistry in Perspective
HIV is an enveloped virus with a cylindrical core within its capsid
The core contains two copies of the (+)-ssRNA, reverse transcriptase, ribonuclease, and integrase
HIV binds to T-4 helper lymphocytes of the immune systemFigure 17J HIV
Structure
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Biochemistry in Perspective
Once bound, HIV fuses with the host cell membrane and releases ssRNA and reverse transcriptase
Immediately makes ssDNA from the viral RNA, which is integrated into the host cell chromosome by the integrase enzyme
Figure 17K Reproductive Cycle of HIV, a Retrovirus
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Biochemistry in Perspective
Proviral components are not activated until the T cell is activated by the immune response
Newly synthesized virus buds from the infected cell
Within hours, an infected host cell’s mRNA has been replaced with viral RNA and the virus has taken over the cell
Figure 17K Reproductive Cycle of HIV, a Retrovirus
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Biochemistry in Perspective
Cell death for infected cells can be triggered by: 1. Viral activation of apoptosis genes 2.Numerous viral particles budding at the same
time 3. Massive viral release, depleting necessary
components 4. Formation of syncytia (large, nonfunctional
multinucleated cell masses) by binding neighboring cells
The timeframe to develop AIDS is 2 to 8 or 10 years
There is no cure for AIDS, although treatments to suppress the virus have led to decreased mortality rates
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Biochemistry in Perspective
Nucleic Acid Methods Techniques used to isolate, purify, and
characterize nucleic acids, use their chemical and physical properties
Use differences in molecular weight or shape, base sequence, or complementary base pairing
Nuclei can be isolated and ruptured to purify the nucleic acid
RNase can remove RNA and DNase can remove the DNA, depending on which nucleic acid you are interested in
Biochemistry in the Lab
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Techniques Adapted from Use with Other Biomolecules—Many techniques used to study proteins can be adapted for use with nucleic acids
Chromatography is extremely useful in protein and DNA purification (ion-exchange chromatography, gel filtration chromatography, and affinity)
Gel electrophoresis is extremely useful to separate DNA by charge (agarose or polyacrylamide)
Biochemistry in the Lab
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Techniques That Exploit the Unique Structural Features of the Nucleic Acids
Denaturation and renaturation of DNA is an important tool in biochemistry and can be visualized with UV spectroscopy (260 nm)
Figure 17D Denaturation and Renaturation of DNA
Biochemistry in the Lab
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
By heating a DNA sample slowly, you can find the melting temperature of that specific DNA molecule (TM)
DNA with high GC content has a higher TM, because of the greater number of hydrogen bonds
Biochemistry in the Lab
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Figure 17E Southern Blotting
Biochemistry in the Lab Southern blotting uses
a complementary hybridization probe and autoradiography to identify targeted DNA fragments
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
Sanger Sequencing
Limited to approximately 1000 nucleotides
Next Generation Sequencing
Next generation sequencing (NGS) is often referred to as massively parallel sequencing, which means that millions of small fragments of DNA can be sequenced at the same time, creating a massive pool of data. This pool of data can reach gigabites in size, which is the equivalent of 1 billion (1,000,000,000) base pairs of DNA. In comparison, previous methods could sequence one DNA fragment at a time, perhaps generating 500 to 1000 base pairs of DNA in a single reaction.
http://www.geneticseducation.nhs.uk/laboratory-process-and-testing-techniques/next-generation-sequencing-ngs
Forensic Investigations DNA can be extracted
from dried biological specimens for many years (e.g., blood, saliva, and hair)
DNA analysis can be used to identify victims or perpetrators of crimes (DNA typing)
DNA fingerprinting was introduced in 1985 and used Southern blotting of minisatellites to compare individuals
Figure 17I Forensic Use of DNA Fingerprinting
Biochemistry in the Lab
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
When DNA quantities are small, they can be amplified with the polymerase chain reaction (PCR)
DNA from a single cell is now sufficient for DNA fingerprint analysis
Figure 17I Forensic Use of DNA Fingerprinting
Biochemistry in the Lab
From McKee and McKee, Biochemistry, 5th Edition, © 2011 Oxford University Press
