1-Nucleotides and Nucleic Acids

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    Powerpoint #1: Nucleotides and Nucleic Acids

    Chemistry and Biochemistry 153B

    DNA, RNA, and Protein Synthesis

    Winter 2016 - A!ert Co"rey 

    UCLA 

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    1.2) Organizing Principle of the Course=Central Dogma of Molecular Biology

    rNPs

    DN! "N! Proteintranscription translation

    Meta#olites$amino aci%s& 'COO(& etc.)

    De noosynthesis

    salagesynthesis

    Brea*%o+n pro%ucts$+aste pro%ucts& useful meta#olites)

    cata#olism%NPs

    processing

    replication

    repair 

    ,ast - ,ectures

    /irst -2 ,ectures

    Why we study this stuff:0 #1 reason$ “the universe enjoys being observed”* and so the "ni%erse

    created creat"res i&e yo" and me 'ho en(oy o!ser%in) the "ni%erse* 0 Other reasons the mechanisms #y +hich genetic information is enco%e%

    an% %eco%e% are central to our un%erstan%ing of +ho +e are as human

    #eings& an% to our un%erstan%ing of human physiology an% human %isease.

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    oet& oet& an% Pratt /igure (

    1.) Coalent 3tructure ofNucleic !ci%s

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     !) ! purine resi%ueB) !n a%enine resi%ue

    C) ! 45 en% resi%ueD) ! %eo6ya%enosine resi%ue7) None of the a#oe

    iClic*er 8uestionTo which of the following things is the

    red arrow NOT pointing?

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    1.9) Nucleoti%e 3tructure

    SugarRibose or Deoxyribose

    BasePurine or Pyrimidine

    Phosphates1, 2, or 3

    Nucleosi%e

    Nucleoti%e

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    1.4) he 3ugars

    /ischerPro:ection

    C

    D(ri#ose,inear al%ehy%e

    ;(D(ri#ose

    $this anomer foun% inri#onucleoti%es an% "N!)

    ;(D(25(%eo6yri#ose$foun% in %eo6ynucleoti%es

    an% DN!)

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    1.?) he Bases

    he #ases in nucleoti%es aresu#stitute% %eriaties of t+o%ifferent heterocycles.

    pyrimidine purine

    hey are classical Bronste% #ases& #ecause N1 an% N of pyrimi%ine& an% N1& N& an%N@ of purine hae pairs of non(#on%ing electrons that can #e use% to form coalent #on%s toprotons. pAa5s are in the 9(4 range.

    8uestions 

    1) ill the nitrogens #e protonate% at physiological p'> hy or +hy not2) hy isn5t N of purine #asic

    :

    : :

    :

    :

    :

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    iClic*er 8uestionWhich of the following statements describes the

    state of the N1 N! and N" nitrogen atoms in purine

    and pyrimidine at physiological pH ? !. hey +ill not #e protonate% #ecause their pAa5s are too high.

    B. hey +ill not #e protonate% #ecause their pAa

    5s are too lo+.

    C. hey +ill all #e protonate%.

    D. Only N@ +ill #e protonate%.

    7. E %on5t *no+.

     

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    iClic*er 8uestionWhy isnt N$ of the purine ring basic?

     !. N is sp hy#ri%ize% an% therefore cannot accept a proton

    B. he non(#on%ing electrons on N are %elocalize%

    C. Protonation of N +oul% lea% to a loss of resonancesta#ilization

    D. Both B an% C

    7. !ll of the a#oe

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    1.F) Purine an% Pyrimi%ine Deriaties in DN! an% "N!

    N

    N

    N

    N

    X

    X

    N

    N

    X

    NH2

    O

    O

    O

    O

    O

    CH3

    N

    NN

    N

    H

    H

    X

    N

    NN

    N

    X

    NH2

    H2N

    H

    O

    C

    G

     !

    H

    E%iosyncratic nomenclature

    12

    9

    4

    ?

    1

    2

    9

    4? @

    F

    Bases Nucleosides Nucleotides

    Cytosine   Cytidine  Cytidylic acid 

    racil   ridine  ridylic acid 

    !hymine   !hymidine  !hymidylic acid 

    "denine   "denosine   "denylic acid 

    #uanine   #uanosine  #uanylic ac i d  

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    1.) Base !romaticity

    • Planarity of aromatic #ases allo+s efficientstac*ing>.  – Because of %elocalize% π or#ital electrons& a%:acently

    stac*e% #ases attract one another strongly throughfaora#le in%uce% %ipole interactions.

     – his stac*ing ma*es a significant contri#ution to the (%imensional structure of nucleic aci%s.

    •  !romatic #ases a#sor# G light. – G spectroscopy can #e use% to pro#e structural transitions

    in nucleic aci%s.

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    1.1I) automerism

    he #ases can e6ist in multiple tautomeric forms.

    N

    N  N

    N

    X

    H2N

    H

    O

    N

    N  N

    N

    X

    H2N

    O

    H

    Aeto tautomer $J.K)

    7nol tautomer LI.1K)

    H

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    iClic*er 8uestion%n the abo&e e'uilibrium why does the (eto

    tautomer predominate o&er the enol tautomer?!. he stan%ar% free energy of the *eto tautomer is greater than

    the stan%ar% free energy of the enol tautomer.

    B. he stan%ar% free energy of the *eto tautomer is less than thestan%ar% free energy of the enol tautomer.

    C. he O(' #on% is more polarize% than the N(' #on%.

    D. he *eto tautomer is #etter a#le to form a atson(Cric*#asepair than is the enol tautomer.

    7. Both B an% C.

    N

    N  N

    N

    X

    H2N

    H

    O

    N

    N  N

    N

    X

    H2N

    OH

    *eto enol

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    1.11) he Phosphates

    pAa = 2 pAa = @ pAa = 12

    • Phosphoric aci% contains three ioniza#le o6ygens

    • Phosphates polymerize #y formation of anhy%ri%e lin*ages

    pyrophosphate

    pAas of pyrophosphoric aci% = 1& 2.4& ?.1& F.4

    Phosphates :oin to sugars #y formation of phosphoester lin*ages

    $e.g.& 25& 5& or 45 O of ri#ose) Monoester pAas 1& ?Diester pAa  1

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    1.12) hat you shoul% *no+ from sli%es 1.(1.11

    E. 3tructures an% nomenclature to *no+ from memory

     !. he #ases sho+n on sli%e 1.F& along +ith theirnucleosi%e an% nucleoti%e %eriaties& inclu%ing atomnum#ering& stereochemistry& an% ionization states.

    B. Primary structure of "N! an% DN! stran%s& inclu%ing

    correct stereochemistry an% ionization states& an% themeaning of stran% polarity. ou shoul% #e a#le torecognize the 5 an% 45 en%s.

    EE. he implications of #ase aromaticity on

     !. Nitrogen #asicityB. he potential for stac*ing

    EEE. Aeto(enol tautomerism ( the relatie sta#ility of thetautomers an% the significance of this to #asepairing

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    )ight *anded anti+parallel

    double heli,

    1.1) atson an% Cric* Mo%el

    81 Base% on a#oe %ata& ho+ many

    #asepairs are there per turn82 Base% on the a#oe %ata& +hat is thehelical pitch

    *elical parameters-

    atson& .D. an% Cric*& /.'.C. $14) Nature1@1& @@(@F&

    hese are the alues that QC %e%uce% from /ran*lin5s fi#er%iffraction %ata. he actual alues for DN! in solution are a little%ifferent. /or e6ample& the aerage helical t+ist in solution ismore li*e 9R.

    Helical rise 3$% &

    Helical twist 3'(Diameter 2) &

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    1.19) "osalin% /ran*lin5s S(ray %iffraction %ata

    /ran*lin& ".'. an% Hosling&

    ".H. $14) Nature 1@1&@9I(@91

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    .raggs /aw-: n  0 dsin .n = an integer λ  = +aelength% = %istance #et+een ro+s of atoms2$θB) = angle of %iffraction that gies rise to constructie interferencehttpQQ+++.matter.org.u*Q%iffractionQgeometryQgeometryTofT%iffractionT#raggsTla+T1.htm

    1.14) Gse of S(ray %iffraction to %etermine molecular %imensions

    x

    ,eft picture constructie interference

    "ight picture %estructie interference

    En phase Out of phase

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    1.1?) hy %o +e use S(rays to %eterminemolecular structure

    0 3o the smallest %istance that can #e measure%#y pro#ing +ith electromagnetic ra%iation is halfthe +aelength.

    0 Coalent #on%s are typically 1(2 U in length.0 3o in or%er to %etermine molecular structures at

    atomic resolution& +e nee% S(rays.

    S(ray crystallographers typically use copper S(ray tu#e $V = 1.491F U)

    2dsinθ B

      = nλ 

    d =nλ 

    2sinθ B

    Smallest possiblevalue for d   =λ 

    2

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    1) ". /ran*lin DN! fi#er S(ray %iffraction %ata

    2) /ran*lin5s %ensity measurements suggeste% a 2 or (stran%e% heli6.) Chargaff5s rules9) W H in *eto tautomeric forms

    1.1@a) Enformation use% #y atson an% Cric* to #uil% their mo%el

    *elical Pitch 0 !2 3

    Cross suggests heli6 ( angle gies helical %iameter of 2I U

    'elical "ise = .9 U/ran*lin& ".'. an% Hosling& ".H.$14) Nature 1@1& @9I(@91

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    1.1@#) Emportance of *eto tautomers

    3li%e 1.1I implies that *eto tautomers pre%ominate oer enoltautomers #y a ratio of J1III to 1. hat is the ei%ence0 Presuma#ly you coul% measure the eXuili#rium ratio #y NM" or some other

    spectroscopic techniXue& #ut E +as una#le to fin% such measurements in theliterature.

    0 atson an% Cric* relie% on Xuantum mechanical calculations to +hich they +erereferre% #y Caen%ish la#oratory colleague erry Donahue.

    0 he *eto tautomeric state has since #een erifie% #y countless crystallographican% NM" stu%ies.

    N

    N  N

    N

    X

    H2N

    H

    O

    N

    N  N

    N

    X

    H2N

    OH

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    1.1@c) 7i%ence that *eto tautomers pre%ominate in io

    Ef or H assume enol form then H #asepairs #ecome possi#le

    0 his coul% result in mismatches %uring DN! replicationan% therefore in mutations.0 he fact that such mutations rarely occur erifies that the

    enol tautomers rarely form in io.

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    iClic*er 8uestionTo calculate the distance 4d5 associated with aspot in a fiber diffraction pattern which of the

    following things do we NOT need to (now?

     !. he %istance of the spot from the center of the %iffraction

    pattern.

    B. he %istance of the S(ray source from the specimen.

    C. he %istance of the film from the specimen.

    D. he +aelength of the ra%iation.

    7. None of the a#oe.

     

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    1 1) t C i * # i

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    1.1) atson(Cric* #asepairsE +ant you to hae a soli% grasp of thesestructures.

    hy Because an un%erstan%ing of thespecificity of #asepairing is critical to anun%erstan%ing of0 Nucleic aci% structure an% chemistry0 DN! replication0 DN! repair 0 ranscription

    0 "N! processing0 ranslation

    %n other words almost e&erything westudy in this course9

     !s +e %iscuss topics li*e DN! replication&"N! processing& an% translation& +e5ll seethat the #asepair rules can sometimes #erela6e%. o un%erstan% ho+ they can #erela6e%& you first nee% to un%erstan% +hat%ictates this specificity in the first place.

    *ence ne,t wee(s 'ui>999oet& oet& an% Pratt/igure 29(1

    iCli * 8 ti

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    iClic*er 8uestionWhy does the NA double heli, only

    contain AT and @ basepairs? !. hese are the only pairs of #ases that hae

    complementary arrays of '(#on%ing groups.

    B. hese are the only pairs of #ases that fit into the %ou#leheli6 +ithout leaing any unsatisfie% '(#on% %onors oracceptors.

    C. hese are the only #asepairs that e6hi#it perfect symmetry.

    D. Both ! an% B.

    7. !ll of the a#oe.

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    1.2I) he pseu%o%ya% a6is

    he glycosi%ic #on%s aresymmetrically arrange%aroun% the pseu%o%ya% a6is.3o the %ou#le heli6 smoothlyaccommo%ates !& !& HC&an% CH #asepairs

    oet& oet& an% Pratt/igure 29(1

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    1.21) hat accounts for the a#sence of non(atson(Cric* #asepairs in DN!

    1 22) h t h l% * f li% 1 1 1 21

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    1.22) hat you shoul% *no+ from sli%es 1.1(1.21E. he atsonQCric* mo%el

     !. hat +as the significance of the follo+ing in formulating the

    mo%el1. "osalin% /ran*lin5s %ata an% Bragg5s la+

    2. Chargaff5s rules

    . Ano+le%ge of the correct tautomeric states

    B. hat are the most important features of the mo%elEE. atsonQCric* #asepairs

     !. ou shoul% #e a#le to %ra+ the ! an% HC #asepairs +ithgood geometry7

    B. hat is the meaning of the pseu%o%ya% a6isC. hy %oes the %ou#le heli6 only accommo%ate these t+o

    #asepairs

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    1 29) Protein Data Ban* $PDB)

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    1.29) Protein Data Ban* $PDB)Molecular graphics programs $such as mol an% Pymol) %ra+ structures of proteins an%nucleic aci%s using %ata in the Protein Data Ban*> $httpQQ+++.rcs#.org).- 7ach structure e6ists as a PDB file& a list of all the atoms +ith their coor%inates.-  !s of 12Q2FQ2I14& 119&4? structures ha% #een %eposite% in the PDB.

    6,perimental ethod0 S(ray structures Y 1I2&1410 NM" structures Y 11&2190 7M structures Y 22

    0 'y#ri% Y F0 Other Y 1

    1 2 ) 7 l f B DN! % ! DN!

    http://www.rcsb.org/http://www.rcsb.org/

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    PDB ED $year) ype of heli6 $length) $resolution) 3eXuence

    1BN! $1F1) B(form $12mer ) $1. U) CHCH!!CHCHB37 $2II) B(form $1?mer ) $1.? U) !C!C!!HHC!!D $2I12) !(form $19mer ) $2.4 U) CCCCHH!CCHHHH1Z/1 $2II4) !(form $1Imer ) $1.4 U) CCHHHCCCHH

    he Dic*erson Do%ecamer> Y the first right(han%e% %ou#le heli6 to haeits structure %etermine% at near atomic resolution

    1.24) 76amples of B(DN! an% !(DN!structures in the PDB

    What does a pdb file loo( li(e? /ets go to the pdb to

    find out7

    1 2?) , t5 P l t l * t B

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    1.2?) ,et5s use Pymol to loo* at B(DN! an% !(DN! structures

    e5ll a%%ress the follo+ing Xuestions1) hat are the ma:or an% minor grooes2) hat %o +e see in the grooes) hat is the position of the helical a6is relatie to the

    #asepairs

    9) hat is the sugar puc*er4) hat %o +e mean #y #asepair tilt?) hat %o +e mean #y propeller t+ist@) hat are the ma:or %ifferences #et+een B(DN! an% !(

    DN!

    e5ll also practice rea%ing the seXuence of DN! #y loo*ing intothe grooes

    http://www.boatdesign.net/forums/attachments/props/19848d1207043400-propellor-pedal-powered-catamaran-cimg0421.jpghttp://www.boatdesign.net/forums/attachments/props/19848d1207043400-propellor-pedal-powered-catamaran-cimg0421.jpg

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    1;BC

    1BC

    'elicala6is

    1.2@) hy are the grooes of %ifferent +i%ths

    1 2F) Differences #et een ! an% B DN!

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    1.2F) Differences #et+een ! an% B(DN!

    %mportant differences

    1) !(DN! has less rise than B(DN! %ue& in part& to the %ifferent sugar puc*ers2) he #ases are shifte% a+ay from helical a6is in !(DN!

    a) "esults in caernous ma:or grooe an% shallo+ minor grooe

    #) "esults in ? U hole

    ) Base pairs %ramatically tilte% in !(DN!

    hese aerages mas* a lot of aria#ilitye.g.& in the Dic*erson Do%ecamer& helicalt+ist aries from 2?R to 9R& +hile helicalrise aries from 2.F U to 9.9 U.

    hile these alues are #ase% on fi#er %iffractionstu%ies& solution stu%ies %emonstrate that hR for B(DN! in solution is -1I.4 #p& correspon%ing to ahelical t+ist of 9R an% a helical pitch of 4.@ U.

    B-DNA A-DNADiameter   20 Å 26 ÅHelical rise   3.4 Å 2.9 Å

    Helical twist   36 *̊ 31˚Helical repeat(h )̊  10* bp 11.6 bpHelical pitch   34 Å* 34 ÅTilt   6 ˚ 20 ˚Propellortwist   11 ˚ 12˚!"arP!c#er   C2’-endo C3’-endo

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    1.2) 7ffect of sugar puc*er on #ac*#one e6tension

    oet& oet& an% Pratt /igures 29(4 an% 29(@

    Butane$Ne+man pro:ection)

    [≅

    14IR $close toanti)

    [ ≅ FIR $close to g()

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    iClic*er 8uestion

    What is this basepair? !) !B) !C) CH

    D) HC7) None of the a#oe

    B37

    N@ O? N9

    O9 N? N@4(me

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    iClic*er 8uestionWhat is this basepair?

     !) !

    B) !C) CHD) HC7) None of the a#oe

    B37

    NO2

    NO2N2

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    1.2) 25(O' group results in steric cro+%ing

    Cro+%ing is +orse in C25 en%o sugar puc*er 

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    iClic*er 8uestionWhy does water stabli>e

    .+NA relati&e to A+NA?

     !) Only the B(DN! minor grooe has appropriate '(#on%inggroups for '(#on%ing to +ater.

    B) he narro+ minor grooe of !(DN! e6clu%es +ater +hile the#roa%er minor grooe of B(DN! a%mits +ater.

    C) Both ! an% B.

    D) None of the a#oe.

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    1.) he atson(Cric* Mo%el

    atson& .D. an% Cric*& /.'.C. $14) Nature1@1& @@(@F&

    1 9) B( s Z(DN!

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    1.9) B( s. Z(DN!

    B(DN!

    B37

    Z(DN!

    2DCH

    1 4) Z s B DN! #asepair

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    1.4) Z( s. B(DN! #asepair 

    syn

    anti

    C5 en%o

    C25 en%o

    anti

    C25 en%o

    anti

    D2DCH

    .1BN!

    stionshy %o #ul*y su#stituents $e.g.& Br) at F position of purine ring faor Z(DN!hy %oes high salt faor Z(DN!

    %f you can answer 'uestion " on P81 you understand e&erything you

    need to (now about D+NA9

    1.?) hat you shoul% *no+ from sli%es 1.2(1.4

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    1.?) hat you shoul% *no+ from sli%es 1.2 1.4E. !( s. B(form helices

     !. hat is the meaning of each of the parameters on sli%e 1.2F

    B. 'o+ %o the oerall shape of !( an% B(form helices %iffer an% ho+%o the %ifferent sugar puc*ers contri#ute to this

    C. hat is the role of +ater in sta#ilizing B( relatie to !(DN!

    D. hat is the role of the 25O' group in sta#ilizing !( relatie to B("N!

    7. 'o+ is the atson(Cric* mo%el a hy#ri% #et+een !( an% B(DN!EE. Z(form helices

     !. hy are Z(form helices restricte% to seXuences that alternate#et+een purine an% pyrimi%ine resi%ues

    B. hat factors influence the sta#ility of Z(form helices

    EEE. ou shoul% #e a#le to rea% the seXuence of a piece of DN! #yloo*ing into the grooes.