nucleic acid lecture

7/30/2019 Nucleic Acid Lecture

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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!


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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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? !


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.!


Lehninger Principles of Biochemistry, Nelson and Cox !C)



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Replication involves unwinding by helicases and DNA synthesis with DNA

polymerases!


Wilson and Walker, Chapter 1, Figure 5.13!&)

. . . is catalyzed by DNA polymerase, ametalloenzyme!



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!



Lehninger Principles of Biochemistry, Nelson and Cox !()

Lehninger, Chapter 26!

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

Transcription: mRNA synthesis!


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Transcription: RNA synthesis!


Wilson and Walker, Chapter 1, Figure 5.14!E)



Wilson and Walker, Chapter 1, Figure 5.15!

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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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. . . !

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9%) Huttenhofer, Trends Gen. 2005, 21, 289.!

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Translation: protein synthesis!


Lehninger Principles of Biochemistry, Nelson and Cox !9C)



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Translation: protein synthesis!


Lehninger Principles of Biochemistry, Nelson and Cox !

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)!9()

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9D) Huttenhofer, Trends Gen. 2005, 21, 289.!

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Lehninger Principles of Biochemistry, Nelson and Cox !9E)



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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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.!


77)


Don#t forget the nomenclature:!




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7%) Lehninger Principles of Biochemistry, Nelson and Cox !

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Nucleic acids are the most structurally flexible biomolecules.!

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Blackburn and Gait, Chapter 2!



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!


7$) Blackburn and Gait, Chapter 2!

Watson-Crick base pairing: hydrogen-bonding interactions!!


cytosine! guanine!



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Watson-Crick base pairing: hydrogen-bonding interactions!!


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! !


7D)

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.!


B form 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!


%8) Wilson and Walker, Chapter 1, Figure 5.5!

B form DNA!




Crick base pairing!







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%9) Wilson and Walker, Chapter 1, Figure 5.5!

B form DNA!




Crick base pairing!






%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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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!


&%G°37 = %G°init + %G°37(GC) + %G°37(CC) + %G°37(CG) + %G¡37(GG) + %G°37(GC) + %G°sym!



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. !


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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9E)


Lehninger Principles of Biochemistry, Nelson and Cox !%()

Transfer RNA (tRNA)!

Lehninger, Chapter 10!



RNA is unstable due to hydrolysis of the phosphodiester!!

%D)



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Lehninger Principles of Biochemistry, Nelson and Cox !%E)



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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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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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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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!

nucleic acid lecture

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