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Genes, Genomes and Nucleic Acid Structure !
Lecture Outline:!
1)Why do we care about Nucleic Acids? Review oftranscription, replication, regulation, etc.!
2)Nucleic Acids: Structure, properties of the nucleicacid polymer, B-form DNA, melting temperature"
3)How do we make DNA for our biochemistry
experiments? DNA synthesis"
9)
Reading: Wilson and Walker, Chapter 5, sections 5.1-5.5!
The Central Dogma of Biology!
Genes, Genomes and Nucleic Acid Structure !
Wilson and Walker, Chapter 1, Figure 5.10!7) Lehninger Principles of Biochemistry, Nelson and Cox !
Genome: Full DNA component that encodes for every protein necessary for the organism!
Proteome: Full protein composition in an organism. !
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7)
Replication: DNA synthesized from DNA!
Transcription: DNA synthesized from RNA!
Translation: proteins synthesized from RNA!
Regulation: turn these events on and off!
Why would we study nucleic acids? !
Genes, Genomes and Nucleic Acid Structure !
Lehninger Principles of Biochemistry, Nelson and Cox !%)
Because they are involved in a wide
variety of biological roles, including: !
We will BRIEFLY review these process- please return to previous
course work to recall these systems, if necessary.!
Replication: DNA synthesis !
A new strand of DNA is synthesized from a DNAtemplate and is the first step in cell mitosis and tissuegrowth.!
Genes, Genomes and Nucleic Acid Structure !
Lehninger Principles of Biochemistry, Nelson and Cox !C)
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Replication: DNA synthesis !
Replication involves unwinding by helicases and DNA synthesis with DNA
polymerases!
Genes, Genomes and Nucleic Acid Structure !
Wilson and Walker, Chapter 1, Figure 5.13!&)
. . . is catalyzed by DNA polymerase, ametalloenzyme!
Replication: DNA synthesis !
Genes, Genomes and Nucleic Acid Structure !
Lehninger Principles of Biochemistry, Nelson and Cox !$)
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How does cell repair the “nick” at the end of DNA synthesis?!
DNA ligase!
Replication: DNA synthesis !
Genes, Genomes and Nucleic Acid Structure !
Lehninger Principles of Biochemistry, Nelson and Cox !()
Lehninger, Chapter 26!
. . . or transcription, is the first step in gene expression!
Transcription: mRNA synthesis!
Genes, Genomes and Nucleic Acid Structure !
D)
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Transcription: RNA synthesis!
Genes, Genomes and Nucleic Acid Structure !
Wilson and Walker, Chapter 1, Figure 5.14!E)
Transcription: RNA synthesis!
Genes, Genomes and Nucleic Acid Structure !
Wilson and Walker, Chapter 1, Figure 5.15!
Typical promoter elements in prokaryotes!
Typical promoter elements in eukaryotes!
98)
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Transcription: RNA synthesis!
Genes, Genomes and Nucleic Acid Structure !
Wilson and Walker, Chapter 1, Figure 5.16!
In prokaryotes, a single promoter can encode for several genes. . .!
99)
Transcription: RNA synthesis!
Genes, Genomes and Nucleic Acid Structure !
Wilson and Walker, Chapter 1, Figure 5.17- 5.18!
In eukaryotes, a single gene can encode for multiple proteins due to post-transcriptionalmodifications, including splicing. . . !
97)
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Genes, Genomes and Nucleic Acid Structure !
9%) Huttenhofer, Trends Gen. 2005, 21, 289.!
F+A4?;+3GHI4)34)G+IJA+BI56?)K6+?/?)6/JA+BI56?)
Translation: protein synthesis!
Genes, Genomes and Nucleic Acid Structure !
Lehninger Principles of Biochemistry, Nelson and Cox !9C)
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Translation: protein synthesis!
Genes, Genomes and Nucleic Acid Structure !
Lehninger Principles of Biochemistry, Nelson and Cox !
tRNA!
L3I;M603?5+B1)L6+N1)FB0I;OJI1)A4>)P5+B6+)
9&)
Regulation is needed to ensure proper functioning of all events. Here is one
example of transcription regulation!
Genes, Genomes and Nucleic Acid Structure !
Wilson and Walker, Chapter 1, Figure 5.16!
In prokaryotes, the lactose (lac) operon is regulated be a repressor protein . . . !
9$)
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Genes, Genomes and Nucleic Acid Structure !
Annunziato,"A."(2008)"DNA packaging: Nucleosomes and chromatin."Nature Education"1(1)!9()
Q4)R/JA+BI56?),),),))
Genes, Genomes and Nucleic Acid Structure !
9D) Huttenhofer, Trends Gen. 2005, 21, 289.!
F+A4?;+3GHI4)34)G+IJA+BI56?)K6+?/?)6/JA+BI56?)
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98)
Replication: DNA synthesized from DNA!
Transcription: DNA synthesized from RNA!
Translation: proteins synthesized from RNA!
Regulation: turn these events on and off!
Why would we study nucleic acids? !
Genes, Genomes and Nucleic Acid Structure !
Lehninger Principles of Biochemistry, Nelson and Cox !9E)
Because they are involved in a wide
variety of biological roles, including: !
How can nucleic acids do all of these diverse functions? !
They are the most flexible biomolecule!!
Polymer consisting of repeating units of nucleotides:!
Nucleic Acid Structure !
Lehninger Principles of Biochemistry, Nelson and Cox !78)
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79)
Each nucleotide is comprised of a purine or pyrimidine base:!
Nucleic Acid Structure !
Wilson and Walker, Chapter 1, Figure 5.1!
The structural unit where a base is bound to a sugar through a glycosidic linkage
is called a nucleoside.!
Lehninger Principles of Biochemistry, Nelson and Cox !
77)
Nucleic Acid Structure !
Don#t forget the nomenclature:!
Wilson and Walker, Chapter 1, Figure 5.1!
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7%) Lehninger Principles of Biochemistry, Nelson and Cox !
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Nucleic Acid Structure !
Nucleic acids are the most structurally flexible biomolecules.!
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Blackburn and Gait, Chapter 2!
Polymer consisting of repeating units of nucleotides:!
Nucleic Acid Structure !
Lehninger Principles of Biochemistry, Nelson and Cox !7C)
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Deoxyribonucleic acids (DNA)!
7&) Wilson and Walker, Chapter 1, Figure 5.3!Blackburn and Gait, Chapter 2!
Deoxyribonucleic acids (DNA)!
7$) Blackburn and Gait, Chapter 2!
Watson-Crick base pairing: hydrogen-bonding interactions!!
Lehninger Principles of Biochemistry, Nelson and Cox !
cytosine! guanine!
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Deoxyribonucleic acids (DNA)!
7()
Watson-Crick base pairing: hydrogen-bonding interactions!!
Wilson and Walker, Chapter 1, Figure 5.4!
Notice that C:G pairing results in
THREE (3) hydrogen bondinginteractions, while A:T pairing
results in only TWO (2) hydrogenbonding interactions. !
The important conclusion is thatC:G base pairings are stronger
than A:T base pairings. !
The differential stability of C:G
versus A:T base pairing will come
into play in various techniqueslater! !
Deoxyribonucleic acids (DNA)!
7D)
Double-stranded DNA can create several different structures.
The most common is B-form double-stranded DNA.!
Wilson and Walker, Chapter 1, Figure 5.5!
B form DNA!
Lehninger Principles of Biochemistry, Nelson and Cox !
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Deoxyribonucleic acids (DNA)!
7E)
B-form double-stranded DNA.!
Wilson and Walker, Chapter 1, Figure 5.5!
B form DNA!
Lehninger Principles of Biochemistry, Nelson and Cox !
The stability of B form DNA is
dictated by several factors:!1)$ Hydrogen bonding in Watson
Crick base pairing!
2)$ Base stacking interactions!
3)$ The hydrophobic effect!
4)$ Charge-charge interactions!
Deoxyribonucleic acids (DNA)!
%8) Wilson and Walker, Chapter 1, Figure 5.5!
B form DNA!
Lehninger Principles of Biochemistry, Nelson and Cox !
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 double-stranded DNA.!
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Deoxyribonucleic acids (DNA)!
%9) Wilson and Walker, Chapter 1, Figure 5.5!
B form DNA!
Lehninger Principles of Biochemistry, Nelson and Cox !
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 double-stranded DNA.!
Deoxyribonucleic acids (DNA)!
%7) Wilson and Walker, Chapter 1, Figure 5.5!Lehninger Principles of Biochemistry, Nelson and Cox !
Practicals! DNA stability is assessing by fluorescence and melting temperatures.!
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Deoxyribonucleic acids (DNA)!
%%)
Practicals! DNA stability is assessing by fluorescence and melting temperatures.!
5#-GCCGGC-3#!3#-CGGCCG-5#!
5#-CGCGCG-3#!3#-GCGCGC-5#!
%G°37 (kcal/mole) !-11.2 ! ! -9.1!TM (°C) ! ! !67.2! ! ! 57.9!
Blackburn and Gait, Chapter 2!
&%G°37 = %G°init + %G°37(GC) + %G°37(CC) + %G°37(CG) + %G¡37(GG) + %G°37(GC) + %G°sym!
Polymer consisting of repeating units of nucleotides:!
Nucleic Acid Structure !
Lehninger Principles of Biochemistry, Nelson and Cox !%C)
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9D)
Ribonucleic Acid (RNA)!
Lehninger Principles of Biochemistry, Nelson and Cox !%&)
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-strandedsegments, reminiscent of proteins. !
Ribonucleic Acid (RNA)!
Lehninger Principles of Biochemistry, Nelson and Cox !%$)
In addition, RNA accomodates
non-Watson-Crick base pairing, or“Wobble” base pairs, more readily
than DNA, because of its diversestructure.!
RNA has a greater versatility than DNA in diversity of conformation!
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Ribonucleic Acid (RNA)!
Lehninger Principles of Biochemistry, Nelson and Cox !%()
Transfer RNA (tRNA)!
Lehninger, Chapter 10!
Ribonucleic Acid (RNA)!
Blackburn and Gait, Chapter 2!
RNA is unstable due to hydrolysis of the phosphodiester!!
%D)
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78)
Replication: DNA synthesized from DNA!
Transcription: DNA synthesized from RNA!
Translation: proteins synthesized from RNA!
Regulation: turn these events on and off!
Why would we study nucleic acids? !
Genes, Genomes and Nucleic Acid Structure !
Lehninger Principles of Biochemistry, Nelson and Cox !%E)
Because they are involved in a wide
variety of biological roles, including: !
With essential roles in all aspects of biology, the study of nucleic acid function
is an active area. But, we need methods to synthesize DNA/RNA for these
studies.!
Depending on the size of the DNA, it can be synthesized in a variety of way:!
1)$ Solid phase nucleic acid synthesis!2)$ Biosynthesis of DNA!
3)$ Polymerase chain reaction (PCR) of DNA!
Lehninger, Chapter 10! Blackburn and Gait, Chapter 3!
Nucleic Acid Synthesis!
C8)
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Lehninger, Chapter 10! Blackburn and Gait, Chapter 3!
Nucleic Acid Synthesis!
C9)
DNA concentration determination!
C7)
http://cdn.idtdna.com/support/technical/TechnicalBulletinPDF/ Oligonucleotide_Yield_Resuspension_and_Storage.pdf!
How long can a synthesized oligo be? The rule of thumb is <60 bases.!
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Lehninger, Chapter 10! Blackburn and Gait, Chapter 3!
DNA concentration determination!
C%)
Lehninger, Chapter 10! Blackburn and Gait, Chapter 3!
DNA concentration determination!
CC)
You have an DNA oligo of the sequence shown below. Calculate the molar
extinction coefficient. Using this value, calculate the concentration of your DNA ifa 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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C&)
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, restrictiondigest analysis."
4)Calculations!
C&)
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!