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Structural features of the DNA(secondary structure)

Double helix; 2 topographic features majorgroove and minor grooveTwo strands antiparallelIn aqueous environment phosphate-sugarbackbone outside of the helix; purine andpyrimidine rings inside the structureStrand stabilized by :H-bonds between complement basesVan der Waals andHydrophobic interactions between stackedbases

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Conformational varieties of thesecondary structure

B-DNA crystallized fromwater; water retained in theinner structure; predominantform under physiologicalconditions10 base pairs/turn of helixRight handedDistance bet base pairs 0.34 nmDiameter 2.0 nm or 20 A

A-DNADehydrated form of B-DNARight handed helix11 base pairs/helixDiameter = 26 A

Z-DNAFound in short stretches ofnative DNA and synthetic DNALeft handed helix12 base pairs/helix

Diameter = 18 A

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Tertiary structure of the DNA

Circular (relaxed) in E.coli; simian virus 40;bacteriophage; certain animal species

Supercoiled DNA extra twisting in the linearduplex; allows DNA to be more compact inthe cell; regulatory role in replication

Topoisomerase change topology of the DNA

Quadruplex DNA four stranded; in protozoanDNA; occur in G-rich regions; regulating andstabilizing telomeres and regulation of geneexpression

Packaging ofeukaryotic DNAintochromosomes

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Management of GeneticInformation

Learning objectives

Understand the mechanism of DNAreplication, RNA synthesis and proteinsynthesis

Understand the basis of genetic manipulationtechnologies

Flow of genetic information DNA replication

A requirement prior to cell divisionIn prokaryotes and eukaryotes

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Replication ineukaryotes

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

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

the leading strand is synthesized continuously inthe 5 -> 3 direction toward the replication forkthe lagging strand is synthesizedsemidiscontinuously (Okazaki fragments) also inthe 5 -> 3 direction, but away from the replicationforklagging strand fragments are joined by theenzyme DNA ligase

Replication fork Enzymes and proteins in DNA replication

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The action of DNA polymerase Start of DNA replication

UnwindingDNA gyraseintroduces a swivelpoint in advance of the replicationforka helicase binds at the replicationfork and promotes unwinding

Replication ineukaryotes

single-stranded binding(SSB) protein protectsexposed regions of single-stranded DNA

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Summary of DNA replication inprokaryotesUnwinding

DNA gyrase introduces a swivel point in advance ofthe replication forka helicase binds at the replication fork and promotesunwindingsingle-stranded binding (SSB) protein protectsexposed regions of single-stranded DNA

Primase catalyzes the synthesis of RNA primerSynthesis

catalyzed by Pol IIIprimer removed by Pol I

DNA ligase seals remaining nicks

Summary of DNA replication inprokaryotes

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

the leading strand is formed continuouslythe lagging strand is formed as a series of Okazakifragments which are later joined

Five DNA polymerases have been found to exist inE. coli

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

Eukaryotic DNA replication

Not as understood as prokaryotic. Due in nosmall 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

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RNA synthesis

TranscriptionTemplate is DNAMajor enzyme: DNA directed RNA polymeraseNo need for primers5 to 3 direction

Requires a promoter region in the template DNAto which the RNA polymerse will bindPromoter several base pairs upstream awayfrom the start site (+1)Termination may be rho factor dependent rhofactor terminates synthesis or rho factorindependent formation of a stable hairpin loop

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Eukarotic transcription have 3 classes of RNA polymerases

RNA pol I transcribes large ribosomal RNAgenesRNA pol II transcribes protein encoding geneRNA pol III transcribes small RNAs (includingtRNA and 50SRNA)

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Post transcriptional modification of theeukaryotic mRNA

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

Summary of Post-transcriptionalmodification ineukaryotic cells

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Protein synthesis

TranslationBased on the m-RNA sequence, geneticcodeStarts from 5 end of the transcriptOccurs in the ribosomesActivation of amino acids attachment to thetRNAInitiation, elongation, termination

Genetic code

Triplet nucleotide one amino acidNonoverlappingNo punctuationDegenerateAlmost universal

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Initiation

Initiation factorsShine-Dalgarno sequence in mRNA30S ribosomeN-formylmet

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Inhibitors of protein synthesis