Sie sind auf Seite 1von 23

!"#$$%&'!"#($%&) *+,)-.

/01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

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!

9)

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!

9)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Genes, Genomes and Nucleic Acid Structure !


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.!

%)

Lehninger Principles of Biochemistry, Nelson and Cox !

Genes, Genomes and Nucleic Acid Structure !


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.!

C)

Lehninger Principles of Biochemistry, Nelson and Cox !

7)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Genes, Genomes and Nucleic Acid Structure !


Replication: DNA synthesis ! Replication involves unwinding by helicases and DNA synthesis with DNA polymerases!

&)

Wilson and Walker, Chapter 1, Figure 5.13!

Genes, Genomes and Nucleic Acid Structure !


Replication: DNA synthesis ! . . . is catalyzed by DNA polymerase, a metalloenzyme!

$)

Lehninger Principles of Biochemistry, Nelson and Cox !

%)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Genes, Genomes and Nucleic Acid Structure !


Replication: DNA synthesis ! How does cell repair the nick at the end of DNA synthesis?!

DNA ligase!

()

Lehninger Principles of Biochemistry, Nelson and Cox !

Genes, Genomes and Nucleic Acid Structure !


Transcription: mRNA synthesis!

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

D)

Lehninger, Chapter 26!

C)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Genes, Genomes and Nucleic Acid Structure !


Transcription: RNA synthesis!

E)

Wilson and Walker, Chapter 1, Figure 5.14!

Genes, Genomes and Nucleic Acid Structure !


Transcription: RNA synthesis!

Typical promoter elements in prokaryotes!

Typical promoter elements in eukaryotes!

98)

Wilson and Walker, Chapter 1, Figure 5.15!

&)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Genes, Genomes and Nucleic Acid Structure !


Transcription: RNA synthesis!
In prokaryotes, a single promoter can encode for several genes. . .!

99)

Wilson and Walker, Chapter 1, Figure 5.16!

Genes, Genomes and Nucleic Acid Structure !


Transcription: RNA synthesis!
In eukaryotes, a single gene can encode for multiple proteins due to post-transcriptional modications, including splicing. . . !

97)

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

$)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Genes, Genomes and Nucleic Acid Structure !


F+A4?;+3GHI4)34)G+IJA+BI56?)K6+?/?)6/JA+BI56?)

9%)

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

Genes, Genomes and Nucleic Acid Structure !


Translation: protein synthesis!

9C)

Lehninger Principles of Biochemistry, Nelson and Cox !

()

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Genes, Genomes and Nucleic Acid Structure !


Translation: protein synthesis!

tRNA!

L3I;M603?5+B1)L6+N1)FB0I;OJI1)A4>)P5+B6+)
9&)

Lehninger Principles of Biochemistry, Nelson and Cox !

Genes, Genomes and Nucleic Acid Structure !


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 . . . !

9$)

Wilson and Walker, Chapter 1, Figure 5.16!

D)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Genes, Genomes and Nucleic Acid Structure !


Q4)R/JA+BI56?),),),))

9()

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

Genes, Genomes and Nucleic Acid Structure !


F+A4?;+3GHI4)34)G+IJA+BI56?)K6+?/?)6/JA+BI56?)

9D)

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

E)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Genes, Genomes and Nucleic Acid Structure !


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!

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

9E)

Lehninger Principles of Biochemistry, Nelson and Cox !

Nucleic Acid Structure !


Polymer consisting of repeating units of nucleotides:!

78)

Lehninger Principles of Biochemistry, Nelson and Cox !

98)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Nucleic Acid Structure !


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 !

Wilson and Walker, Chapter 1, Figure 5.1!

Nucleic Acid Structure !


Don#t forget the nomenclature:!

77)

Wilson and Walker, Chapter 1, Figure 5.1!

99)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Nucleic Acid Structure !


Nucleic acids are the most structurally exible biomolecules.!

O P ! O " # % O & P O
7%)

(2 O $ (1 (0 (4

(3 '
N

X
)*+,%&+(-+&.( !"#$%&'(

Blackburn and Gait, Chapter 2!

Lehninger Principles of Biochemistry, Nelson and Cox !

Nucleic Acid Structure !


Polymer consisting of repeating units of nucleotides:!

7C)

Lehninger Principles of Biochemistry, Nelson and Cox !

97)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Deoxyribonucleic acids (DNA)!

7&)

Blackburn and Gait, Chapter 2!

Wilson and Walker, Chapter 1, Figure 5.3!

Deoxyribonucleic acids (DNA)!


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

cytosine!
7$)

guanine!
Lehninger Principles of Biochemistry, Nelson and Cox !

Blackburn and Gait, Chapter 2!

9%)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Deoxyribonucleic acids (DNA)!


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! !

7()

Wilson and Walker, Chapter 1, Figure 5.4!

Deoxyribonucleic acids (DNA)!


Double-stranded DNA can create several different structures. The most common is B-form double-stranded DNA.!

B form DNA!
7D)

Lehninger Principles of Biochemistry, Nelson and Cox !

Wilson and Walker, Chapter 1, Figure 5.5!

9C)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Deoxyribonucleic acids (DNA)!


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!
7E)

Lehninger Principles of Biochemistry, Nelson and Cox !

Wilson and Walker, Chapter 1, Figure 5.5!

Deoxyribonucleic acids (DNA)!


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!
%8)

Lehninger Principles of Biochemistry, Nelson and Cox !

Wilson and Walker, Chapter 1, Figure 5.5!

9&)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Deoxyribonucleic acids (DNA)!


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!
%9)

Lehninger Principles of Biochemistry, Nelson and Cox !

Wilson and Walker, Chapter 1, Figure 5.5!

Deoxyribonucleic acids (DNA)!


Practicals! DNA stability is assessing by uorescence and melting temperatures.!

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

Wilson and Walker, Chapter 1, Figure 5.5!

9$)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Deoxyribonucleic acids (DNA)!


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!


%%)

Blackburn and Gait, Chapter 2!

Nucleic Acid Structure !


Polymer consisting of repeating units of nucleotides:!

%C)

Lehninger Principles of Biochemistry, Nelson and Cox !

9()

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

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. !

%&)

Lehninger Principles of Biochemistry, Nelson and Cox !

Ribonucleic Acid (RNA)!


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.!

%$)

Lehninger Principles of Biochemistry, Nelson and Cox !

9D)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Ribonucleic Acid (RNA)!


Transfer RNA (tRNA)!

%()

Lehninger Principles of Biochemistry, Nelson and Cox !

Ribonucleic Acid (RNA)!


RNA is unstable due to hydrolysis of the phosphodiester!!

%D) Lehninger, Chapter 10!

Blackburn and Gait, Chapter 2!

9E)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Genes, Genomes and Nucleic Acid Structure !


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!

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!
%E)

Lehninger Principles of Biochemistry, Nelson and Cox !

Nucleic Acid Synthesis!

C8)

Lehninger, Chapter 10!

Blackburn and Gait, Chapter 3!

78)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

Nucleic Acid Synthesis!

C9)

Lehninger, Chapter 10!

Blackburn and Gait, Chapter 3!

DNA concentration determination!


How long can a synthesized oligo be? The rule of thumb is <60 bases.!

C7)

http://cdn.idtdna.com/support/technical/TechnicalBulletinPDF/ Oligonucleotide_Yield_Resuspension_and_Storage.pdf!

79)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

DNA concentration determination!

C%)

Lehninger, Chapter 10!

Blackburn and Gait, Chapter 3!

DNA concentration determination!


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# !

CC)

Lehninger, Chapter 10!

Blackburn and Gait, Chapter 3!

77)

!"#$$%&'!"#($%&) *+,)-./01)23456+)7899)

:/;<63;)=;3>?) @A4/A+B)7&1)7899)

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!
C&)

7%)