Nucleic Acid Lecture

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Genes, Genomes and Nucleic Acid Structure ! Lecture Outline:! 1)! Why do we care about Nucleic Acids? Review of transcription, replication, regulation, etc.! 2)! Nucleic Acids: Structure, properties of the nucleic acid polymer, B-form DNA, melting temperature" 3)! How do we make DNA for our biochemistry experiments? DNA synthesis"
Reading: Wilson and Walker, Chapter 5, sections 5.1-5.5!
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Genes, Genomes and Nucleic Acid Structure !

The Central Dogma of Biology!
Genome: Full DNA component that encodes for every protein necessary for the organism! Proteome: Full protein composition in an organism. !
7) Lehninger Principles of Biochemistry, Nelson and Cox !
Wilson and Walker, Chapter 1, Figure 5.10!
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Why would we study nucleic acids? ! Because they are involved in a wide variety of biological roles, including: !
Replication: DNA synthesized from DNA! Transcription: DNA synthesized from RNA! Translation: proteins synthesized from RNA! Regulation: turn these events on and off!
We will BRIEFLY review these process- please return to previous course work to recall these systems, if necessary.!
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Lehninger Principles of Biochemistry, Nelson and Cox !

Replication: DNA synthesis ! A new strand of DNA is synthesized from a DNA template and is the rst step in cell mitosis and tissue growth.!
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Replication: DNA synthesis ! Replication involves unwinding by helicases and DNA synthesis with DNA polymerases!
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Replication: DNA synthesis ! . . . is catalyzed by DNA polymerase, a metalloenzyme!
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Replication: DNA synthesis ! How does cell repair the nick at the end of DNA synthesis?!
DNA ligase!
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Transcription: mRNA synthesis!
. . . or transcription, is the rst step in gene expression!
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Lehninger, Chapter 26!
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Transcription: RNA synthesis!
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Typical promoter elements in prokaryotes!
Typical promoter elements in eukaryotes!
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In prokaryotes, a single promoter can encode for several genes. . .!
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In eukaryotes, a single gene can encode for multiple proteins due to post-transcriptional modications, including splicing. . . !
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Wilson and Walker, Chapter 1, Figure 5.17- 5.18!
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Huttenhofer, Trends Gen. 2005, 21, 289.!

Translation: protein synthesis!
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Translation: protein synthesis!
tRNA!
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Regulation is needed to ensure proper functioning of all events. Here is one example of transcription regulation!
In prokaryotes, the lactose (lac) operon is regulated be a repressor protein . . . !
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Annunziato,"A."(2008)"DNA packaging: Nucleosomes and chromatin."Nature Education"1(1)!

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How can nucleic acids do all of these diverse functions? ! They are the most exible biomolecule!!
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Nucleic Acid Structure !

Polymer consisting of repeating units of nucleotides:!
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Each nucleotide is comprised of a purine or pyrimidine base:!
The structural unit where a base is bound to a sugar through a glycosidic linkage is called a nucleoside.!
79) Lehninger Principles of Biochemistry, Nelson and Cox !

Don#t forget the nomenclature:!
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Nucleic acids are the most structurally exible biomolecules.!
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Deoxyribonucleic acids (DNA)!
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Watson-Crick base pairing: hydrogen-bonding interactions!!
cytosine!
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guanine!
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Watson-Crick base pairing: hydrogen-bonding interactions!!
Notice that C:G pairing results in THREE (3) hydrogen bonding interactions, while A:T pairing results in only TWO (2) hydrogen bonding interactions. ! The important conclusion is that C:G base pairings are stronger than A:T base pairings. ! The differential stability of C:G versus A:T base pairing will come into play in various techniques later! !
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Double-stranded DNA can create several different structures. The most common is B-form double-stranded DNA.!
B form DNA!
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B-form double-stranded DNA.! The stability of B form DNA is dictated by several factors:! 1)$ Hydrogen bonding in Watson Crick base pairing! 2)$ Base stacking interactions! 3)$ The hydrophobic effect! 4)$ Charge-charge interactions!
B form DNA!
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B form DNA!
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B form DNA!
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Practicals! DNA stability is assessing by uorescence and melting temperatures.!
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Practicals! DNA stability is assessing by uorescence and melting temperatures.!
5#-GCCGGC-3#! 3#-CGGCCG-5#! %G37 (kcal/mole) !-11.2 ! TM (C) ! ! !67.2! ! ! ! !
5#-CGCGCG-3#! 3#-GCGCGC-5#! -9.1! 57.9!
&%G37 = %Ginit + %G37(GC) + %G37(CC) + %G37(CG) + %G37(GG) + %G37(GC) + %Gsym!

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Ribonucleic Acid (RNA)!

RNA has a greater versatility than DNA in diversity of conformation!
RNA can form double-stranded structures, like DNA, but can also form globular structures comprised of double-stranded and single-stranded segments, reminiscent of proteins. !
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RNA has a greater versatility than DNA in diversity of conformation!
In addition, RNA accomodates non-Watson-Crick base pairing, or Wobble base pairs, more readily than DNA, because of its diverse structure.!
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Transfer RNA (tRNA)!
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RNA is unstable due to hydrolysis of the phosphodiester!!
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With essential roles in all aspects of biology, the study of nucleic acid function is an active area. But, we need methods to synthesize DNA/RNA for these studies.! Depending on the size of the DNA, it can be synthesized in a variety of way:! 1)$ Solid phase nucleic acid synthesis! 2)$ Biosynthesis of DNA! 3)$ Polymerase chain reaction (PCR) of DNA!
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Nucleic Acid Synthesis!
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Nucleic Acid Synthesis!
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DNA concentration determination!

How long can a synthesized oligo be? The rule of thumb is <60 bases.!
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http://cdn.idtdna.com/support/technical/TechnicalBulletinPDF/ Oligonucleotide_Yield_Resuspension_and_Storage.pdf!
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You have an DNA oligo of the sequence shown below. Calculate the molar extinction coefcient. Using this value, calculate the concentration of your DNA if a 1:20 dilution of your stock solution gives an Absorbance of 0.805 at 260 nm using a cuvette with 1 cm pathlength.! M13 forward sequencing primer: 5#-GTAAAACGACGGCCAGTG-3# !
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Working with Bacterial and Mammalian Cells! Lecture Outline:! 1)! Working with Mammalian cells: common cell lines; sterile technique and growth conditions! 2)! Working with Bacteria: bacterial growth and media; quantitative monitoring of cell growth; use of antibiotics; sterile technique! 3)! Plasmid DNA: plasmid map, bacterial transformation, isolation of plasmid DNA from bacteria, restriction digest analysis." 4)! Calculations! Reading: Wilson and Walker, Chapter 2, sections 2.1-2.5 and 2.7 and Chapter 5, sections 5.6-5.7 and 5.9!
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Nucleic Acid Lecture

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Reading: Wilson and Walker, Chapter 5, sections 5.1-5.5!

Genes, Genomes and Nucleic Acid Structure !

7) Lehninger Principles of Biochemistry, Nelson and Cox !

Wilson and Walker, Chapter 1, Figure 5.10!

Genes, Genomes and Nucleic Acid Structure !

Lehninger Principles of Biochemistry, Nelson and Cox !

Genes, Genomes and Nucleic Acid Structure !

Lehninger Principles of Biochemistry, Nelson and Cox !

Genes, Genomes and Nucleic Acid Structure !

Wilson and Walker, Chapter 1, Figure 5.13!

Genes, Genomes and Nucleic Acid Structure !

Lehninger Principles of Biochemistry, Nelson and Cox !

Genes, Genomes and Nucleic Acid Structure !

Lehninger Principles of Biochemistry, Nelson and Cox !

Genes, Genomes and Nucleic Acid Structure !

. . . or transcription, is the rst step in gene expression!

Lehninger, Chapter 26!

Genes, Genomes and Nucleic Acid Structure !

Wilson and Walker, Chapter 1, Figure 5.14!

Genes, Genomes and Nucleic Acid Structure !

Typical promoter elements in prokaryotes!

Typical promoter elements in eukaryotes!

Wilson and Walker, Chapter 1, Figure 5.15!

Genes, Genomes and Nucleic Acid Structure !

Wilson and Walker, Chapter 1, Figure 5.16!

Genes, Genomes and Nucleic Acid Structure !

Wilson and Walker, Chapter 1, Figure 5.17- 5.18!

Genes, Genomes and Nucleic Acid Structure !

Huttenhofer, Trends Gen. 2005, 21, 289.!

Genes, Genomes and Nucleic Acid Structure !

Lehninger Principles of Biochemistry, Nelson and Cox !

Genes, Genomes and Nucleic Acid Structure !

Lehninger Principles of Biochemistry, Nelson and Cox !

Genes, Genomes and Nucleic Acid Structure !

Wilson and Walker, Chapter 1, Figure 5.16!

Genes, Genomes and Nucleic Acid Structure !

Annunziato,"A."(2008)"DNA packaging: Nucleosomes and chromatin."Nature Education"1(1)!

Genes, Genomes and Nucleic Acid Structure !

Huttenhofer, Trends Gen. 2005, 21, 289.!

Genes, Genomes and Nucleic Acid Structure !

Lehninger Principles of Biochemistry, Nelson and Cox !

Nucleic Acid Structure !

Lehninger Principles of Biochemistry, Nelson and Cox !

Nucleic Acid Structure !

79) Lehninger Principles of Biochemistry, Nelson and Cox !

Wilson and Walker, Chapter 1, Figure 5.1!

Nucleic Acid Structure !

Wilson and Walker, Chapter 1, Figure 5.1!

Nucleic Acid Structure !

Blackburn and Gait, Chapter 2!

Lehninger Principles of Biochemistry, Nelson and Cox !

Nucleic Acid Structure !

Lehninger Principles of Biochemistry, Nelson and Cox !

Deoxyribonucleic acids (DNA)!

Blackburn and Gait, Chapter 2!

Wilson and Walker, Chapter 1, Figure 5.3!

Deoxyribonucleic acids (DNA)!

Blackburn and Gait, Chapter 2!

Deoxyribonucleic acids (DNA)!

Wilson and Walker, Chapter 1, Figure 5.4!

Deoxyribonucleic acids (DNA)!

Lehninger Principles of Biochemistry, Nelson and Cox !

Wilson and Walker, Chapter 1, Figure 5.5!