7/21/2011

Learning objectives

Nucleic Acid Chemistry, Structure, and Functions

Understand the relationships between purines, pyrimidines, nucleosides, nucleotides, and nucleic acids. Understand the molecular structure of DNA.

Nucleic acid functions

DNA genetic information storehouse RNA

message between the genome and the ribosome for the building of proteins (mRNA), act as the catalytic moiety in a RNA-protein enzyme complex (rRNA) serve as the translator between mRNA sequence and protein sequence (tRNA)

9000 year old Kennewick man skull discovered along the banks of the Columbia River in Washington state believed to have originated from Southeast Asia, Japan or Polynesia DNA from archeological samples Compared to modern DNA Evidence of DNA stability

RNA and DNA- recent discovery catalyze some reactions without protein involvement (ribozymes or deoxyribozymes).

7/21/2011

Nucleotide monomeric unit in nucleic acid

7/21/2011

7/21/2011

Structural features of the DNA (secondary structure)

Double helix; 2 topographic features major groove and minor groove Two strands antiparallel In aqueous environment phosphate-sugar backbone outside of the helix; purine and pyrimidine rings inside the structure Strand stabilized by : H-bonds between complement bases Van der Waals and Hydrophobic interactions between stacked bases

7/21/2011

Conformational varieties of the secondary structure

B-DNA crystallized from water; water retained in the inner structure; predominant form under physiological conditions
10 base pairs/turn of helix Right handed Distance bet base pairs 0.34 nm Diameter 2.0 nm or 20 A

A-DNA
Dehydrated form of B-DNA Right handed helix 11 base pairs/helix Diameter = 26 A

Z-DNA

Found in short stretches of native DNA and synthetic DNA Left handed helix 12 base pairs/helix Diameter = 18 A

7/21/2011

Geometry attribute:
Helix sense Rotation/bp

A-form

B-form

Z-form

right-handed right-handed left-handed 33.6 35.9 10.5 3.4 (0.34 nm) 34 (3.32 nm) 60/2 12 3.7 (0.37 nm) 45.6 (4.56 nm)

Mean bp/turn 11 Rise/bp along 2.4 axis (0.24 nm) Rise/turn of helix Diameter 24.6 (2.46 nm)

26 (2.6 nm) 20 (2.0 nm) 18 (1.8 nm)

85-90 degrees as the melting temperature

Tm = midpoint of each curve

7/21/2011

Tertiary structure of the DNA

Circular (relaxed) in E.coli; simian virus 40; bacteriophage; certain animal species Supercoiled DNA extra twisting in the linear duplex; allows DNA to be more compact in the cell; regulatory role in replication

Topoisomerase change topology of the DNA

Quadruplex DNA four stranded; in protozoan DNA; occur in G-rich regions; regulating and stabilizing telomeres and regulation of gene expression

Packaging of eukaryotic DNA into chromosomes

7/21/2011

Learning objectives

Management of Genetic Information

Understand the mechanism of DNA replication, RNA synthesis and protein synthesis Understand the basis of genetic manipulation technologies

Flow of genetic information

DNA replication

A requirement prior to cell division In prokaryotes and eukaryotes

7/21/2011

Two possible models of the DNA replication

Expt by Meselson-Stahl proved the semiconservative model of replication

Which direction does replication go?

Replication in prokaryotes

Major enzyme: DNA polymerase III

DNA double helix unwinds at a specific point called an origin of replication Polynucleotide chains are synthesized in both directions from the origin of replication; DNA replication is bidirectional in most organisms At each origin of replication, there are two replication forks, points at which new polynucleotide chains are formed There is one origin of replication and two replication forks in the circular DNA of prokaryotes In replication of a eukaryotic chromosome, there are several origins of replication and two replication forks at each origin

7/21/2011

Replication in eukaryotes

DNA synthesis based on two template strands: leading strand and lagging strand templates; mechanism in prokaryotes is presented

DNA is synthesized from its 5 -> 3 end (from the 3 -> 5 direction of the template)

the leading strand is synthesized continuously in the 5 -> 3 direction toward the replication fork the lagging strand is synthesized semidiscontinuously (Okazaki fragments) also in the 5 -> 3 direction, but away from the replication fork lagging strand fragments are joined by the enzyme DNA ligase

Replication fork

Enzymes and proteins in DNA replication

10

7/21/2011

The action of DNA polymerase

Start of DNA replication

Unwinding DNA gyrase introduces a swivel point in advance of the replication fork a helicase binds at the replication fork and promotes unwinding

Replication in eukaryotes

single-stranded binding (SSB) protein protects exposed regions of singlestranded DNA

11

7/21/2011

Primase catalyzes the synthesis of RNA primer Synthesis

12

7/21/2011

Summary of DNA replication in prokaryotes Unwinding

DNA gyrase introduces a swivel point in advance of the replication fork a helicase binds at the replication fork and promotes unwinding single-stranded binding (SSB) protein protects exposed regions of single-stranded DNA Primase catalyzes the synthesis of RNA primer Synthesis catalyzed by Pol III primer removed by Pol I DNA ligase seals remaining nicks

Summary of DNA replication in prokaryotes

DNA synthesis is bidirectional DNA synthesis is in the 5 -> 3 direction

the leading strand is formed continuously the lagging strand is formed as a series of Okazaki fragments which are later joined

Five DNA polymerases have been found to exist in E. coli

Pol I is involved in synthesis and repair Pol II, IV, and V are for repair under unique conditions Pol III is primarily responsible for synthesis

Eukaryotic DNA replication

Not as understood as prokaryotic. Due in no small part to higher level of complexity. Cell growth and division divided into phases: M, G1, S, and G2 DNA replication occurs during the S phase

13

7/21/2011

RNA synthesis

Transcription Template is DNA Major enzyme: DNA directed RNA polymerase No need for primers 5 to 3 direction

Requires a promoter region in the template DNA to which the RNA polymerse will bind Promoter several base pairs upstream away from the start site (+1) Termination may be rho factor dependent rho factor terminates synthesis or rho factor independent formation of a stable hairpin loop

14

7/21/2011

Eukarotic transcription have 3 classes of RNA polymerases

RNA pol I transcribes large ribosomal RNA genes RNA pol II transcribes protein encoding gene RNA pol III transcribes small RNAs (including tRNA and 50SRNA)

15

7/21/2011

Post transcriptional modification of the eukaryotic mRNA

Capping methyl guanosine attachment at the 5 end to protect the cleavage of the RNA by exonucleases as RNA moves out of the nucleus Addition of poly A at the 3 end (20-250 long) helps to stabilize the mRNA structure; increases resistance to cellular nucleases Splicing removal of non coding sequences (introns)

Summary of Post-transcriptional modification in

eukaryotic cells

16

7/21/2011

Protein synthesis

Genetic code

Translation Based on the m-RNA sequence, genetic code Starts from 5 end of the transcript Occurs in the ribosomes Activation of amino acids attachment to the tRNA Initiation, elongation, termination

Triplet nucleotide one amino acid Nonoverlapping No punctuation Degenerate Almost universal

17

7/21/2011

Initiation

Initiation factors Shine-Dalgarno sequence in mRNA 30S ribosome N-formylmet

18

7/21/2011

Inhibitors of protein synthesis

19