Biochemistry 2/e - Garrett & Grisham

Chapter 12
Structure of Nucleic Acids
to accompany Biochemistry, 2/e by Reginald Garrett and harles Grisham
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Biochemistry 2/e - Garrett & Grisham

Outline
! ! ! ! ! ! ! "2#" $rimary Structure of Nucleic Acids "2#2 AB%s of &NA Secondary Structure "2#' &enaturation and Renaturation of &NA "2#( )ertiary Structure of &NA "2#* hromosome Structure "2#+ hemical Synthesis of Nucleic Acids "2#, Secondary and )ertiary Structure of RNA

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Biochemistry 2/e - Garrett & Grisham

Copyright © 1999 by Harcourt Brace & Company

dideo/y method0.Biochemistry 2/e .4-ray film de1elops in response to presence of radioacti1e isotopes in nucleic acid molecules Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham Primary Structure Se-uencing Nucleic Acids ! hain termination method . de1eloped by 2# Sanger ! Base-specific chemical clea1age. de1eloped by 3a/am and Gilbert ! Both use autoradiography .

Biochemistry 2/e .Garrett & Grisham DNA Replication ! &NA is a double-helical molecule ! 5ach strand of the heli/ must be copied in complementary fashion by &NA polymerase ! 5ach strand is a template for copying ! &NA polymerase re-uires template and primer ! $rimer6 an oligonucleotide that pairs 7ith the end of the template molecule to form ds&NA ! &NA polymerases add nucleotides in *8-'8 direction Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

ddG)$. dG)$. one of 7hich is $-'2-labelled ! 5ach reaction also contains a small amount of one dideo/ynucleotide6 either ddA)$. dd )$ or dd))$ Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham Chain Termination Method Based on DNA polymerase reaction ! Run four separate reactions ! 5ach reaction mi/ture contains dA)$. d )$ and d))$.Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Chain Termination Method ! 3ost of the time.optimally0 at least a fe7 chains terminated at e1ery occurrence of a gi1en nucleotide Copyright © 1999 by Harcourt Brace & Company . the polymerase uses normal nucleotides and &NA molecules gro7 normally ! 9ccasionally. the polymerase uses a dideo/ynucleotide. 7hich adds to the chain and then pre1ents further gro7th in that molecule ! Random insertion of dd-nucleotides lea1es .

Biochemistry 2/e .read *8 to '8 Copyright © 1999 by Harcourt Brace & Company . long fragments on top ! Read the :se-uence: from bottom of gel to top ! on1ert this :se-uence: to the complementary se-uence ! No7 read from the other end and you ha1e the se-uence you 7anted .Garrett & Grisham Chain Termination Method ! Run each reaction mi/ture on electrophoresis gel ! Short fragments go to bottom.

each clea1ed at .Garrett & Grisham Chemical Cleava e Method ! ! ! Not used as !re"uently as San er#s Start 7ith ss&NA labelled 7ith $-'2 at one end Strand is clea1ed by chemical reagents Assumption is that strands of all possible lengths.Biochemistry 2/e . 7ill be produced# 2ragments are electrophoresed and se-uence is read ! Copyright © 1999 by Harcourt Brace & Company .ust one of the occurrences of a gi1en base.

Garrett & Grisham Chemical Cleava e Method ! ! ! ! $our reactions are used G-specific clea1age 7ith dimethyl sulfate.hydra<ine and piperidine0. follo7ed by strand scission 7ith piperidine G/A clea1age6 depurination 7ith mild acid. but high salt protects ) residues Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e . follo7ed by piperidine /) clea1age6 ring hydrolysis by hydra<ine. follo7ed by piperidine clea1age6 same method .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

*8 to '80 Note that the nucleotide closest to the $-'2 7ill be missed in this procedure Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham Chemical Cleava e Method ! ! ! ! Readin the els%%% =t depends on 7hich end of the ss&NA 7as radioacti1ely labelled> =f the *8-end 7as labelled.*8 to '80 =f the '8-end 7as labelled. read the se-uence from bottom of gel to top . read the se-uence from top of gel to bottom .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

hydrogen-bonded0 inside Right-t7ist closes the gaps bet7een base pairs to '#( A .?#'( nm0 in B-&NA Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .Garrett & Grisham The AB&s o! DNA ! ! ! ! Secondary Structure See 2igure "2#"? for details of &NA secondary structure Sugar-phosphate bac@bone outside Bases .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham The 'canonical( )ase pairs ! ! ! ! ! See $i ure 12%1* )he canonical A6) and G6 base pairs ha1e nearly identical o1erall dimensions A and ) share t7o A-bonds G and share three A-bonds G6 -rich regions of &NA are more stable $olar atoms in the sugar-phosphate bac@bone also form A-bonds Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

or groo1e ! )he ma. 12%11 ! )he :tops: of the bases .transcription factors0 can recogni<e the pattern of bases and Abonding possibilities in the ma.or groo1e is large enough to accommodate an alpha heli/ from a protein ! Regulatory proteins .Biochemistry 2/e .Garrett & Grisham Ma+or and minor rooves See $i ures 12%1*.as 7e dra7 them0 line the :floor: of the ma.or groo1e Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e - Garrett & Grisham

Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham

Comparison o! A, B, & DNA
! ! ! ! See Ta)le 12%1 A6 right-handed, short and broad, 2#' A, "" bp per turn B6 right-handed, longer, thinner, '#'2 A, "? bp per turn %6 left-handed, longest, thinnest, '#B A, "2 bp per turn See 2igure "2#"'

Copyright © 1999 by Harcourt Brace & Company

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Rich 2ound in G6 -rich regions of &NA G goes to syn conformation stays anti but 7hole nucleoside .Biochemistry 2/e .base and sugar0 flips "B? degrees Result is that G6 A-bonds can be preser1ed in the transition from B-form to %-form> Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham &-DNA ! ! ! ! Discovered )y Ale.

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

reflecting the re-establishment of stac@ing Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e . its EF absorbance increases by '?-(?G )his hyperchromic shift reflects the un7inding of the &NA double heli/ Stac@ed base pairs in nati1e &NA absorb less light Chen ) is lo7ered. Chen &NA is heated to B?D degrees elsius. the absorbance drops.Garrett & Grisham 12%/ Denaturation o! DNA ! ! ! ! See 2igure "2#".

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

ten bp per turn of heli/ ! ircular &NA sometimes has more or less than "? bp per turn .Biochemistry 2/e .Garrett & Grisham 12%0 Supercoils and Cruci!orms ! =n duple/ &NA.a supercoiled state ! 5n<ymes called topoisomerases or gyrases can introduce or remo1e supercoils ! ruciforms occur in palindromic regions of &NA ! Negati1e supercoiling may promote cruciforms Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Chromosome Structure ! Auman &NAHs total length is I2 meters> ! )his must be pac@aged into a nucleus that is about * micrometers in diameter ! )his represents a compression of more than "??.Biochemistry 2/e .???> ! =t is made possible by 7rapping the &NA around protein spools called nucleosomes and then pac@ing these in helical filaments Copyright © 1999 by Harcourt Brace & Company .

the nucleoprotein comple/.Biochemistry 2/e .Garrett & Grisham Nucleosome Structure ! hromatin. and 7ith &NA by Richmond0 ! Nonhistone proteins are regulators of gene e/pression Copyright © 1999 by Harcourt Brace & Company .7ithout &NA by 3oudriana@is. consists of histones and nonhistone chromosomal proteins ! Aistone octamer structure has been sol1ed .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Chemical Synthesis o! Nucleic Acids ! Jaboratory synthesis of nucleic acids re-uires comple/ strategies ! 2unctional groups on the monomeric units are reacti1e and must be bloc@ed ! orrect phosphodiester lin@ages must be made ! Reco1ery at each step must high> Copyright © 1999 by Harcourt Brace & Company .

second base is added as a nucleoside phosphoramidate Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham Solid Phase Oli onucleotide Synthesis ! &imetho/ytrityl group bloc@s the *H-9A of the first nucleoside 7hile it is lin@ed to a solid support by the 'H-9A ! Step "6 &etritylation by trichloroacetic acid e/poses the *H-9A ! Step 26 =n coupling reaction.Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

and more stable. phosphate group Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .Garrett & Grisham Solid Phase Synthesis ! Step '6 capping 7ith acetic anhydride bloc@s unreacted *H-9As of N-" from further reaction ! Step (6 $hosphite lin@age bet7een N-" and N-2 is reacti1e and is o/idi<ed by a-ueous iodine to form the desired.

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

alone0 is @no7n $henylalanine tRNA is :J-shaped: 3any non-canonical base pairs found in tRNA ! ! ! Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham 12%1 Sec2Tert Structure o! RNA ! Trans!er RNA 5/tensi1e A-bonding creates four double helical domains. three capped by loops. one by a stem 9nly one tRNA structure .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e .assumed0 e/tensi1e base-pairing Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham Ri)osomal RNA Ri)osomes synthesi3e proteins ! All ribosomes contain large and small subunits ! rRNA molecules ma@e up about 2/' of ribosome ! Aigh intrastrand se-uence complementarity leads to .

Biochemistry 2/e .Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .

Biochemistry 2/e . 7hereas se-uence is not ! )here must be common designs and functions that must be conser1ed Copyright © 1999 by Harcourt Brace & Company .Garrett & Grisham Ri)osomal RNA ! Secondary structure features seem to be conser1ed.

Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company .Biochemistry 2/e .