Biochemistry 462a Nucleic Acid Structure Reading - Chapter 10 Practice problems - Chapter 10: #2,3,6,8,9,11; Nucleic Acids extra problems

Transcription

1 Bichemistry 462a ucleic Acid Structure Reading - Chapter 10 Practice prblems - Chapter 10: #2,3,6,8,9,11; ucleic Acids extra prblems Primary Structure As is the case fr prteins, nucleic acids have a primary structure -the sequence f bases alng the plymer - and directinality with a free 5 hydrxyl at ne end and a free 3 hydrxyl at the ther end. By cnventin, nucleic acid sequences are written starting frm the free 5 hydrxyl and ending at the free 3 hydrxyl. In this cnventin, the phsphdiester bnds run 3 t 5. Secndary Structure The secndary structure f DA is the well-knwn duble helix. This structure was deduced frm the analysis f the x-ray diffractin pattern f fibers f DA. Fiber diffractin patterns d nt cntain the mlecular infrmatin fund in single crystal diffractin patterns, but the fiber diffractin patterns and the prperties f the fibers prvided the essential infrmatin t deduce the duble helix structure: The diffractin pattern shwed the presence f a helix. The spacing between the spts shwed that there are 10 bases per turn f the helix. The density f the fibers indicated tw mlecules f DA per helix. Watsn and Crick prpsed that the duble helix was stabilized by hydrgen bnding between bases n the ppsite strands - A T and G C. MAJR GRVE MAJR GRVE C 3 dexyribse A T dexyribse dexyribse G C deyribse MIR GRVE A-T base pair. MIR GRVE G-C base pair In their mdel the hydrphilic sugar-phsphate backbne are n the utside, in cntact with water, and the hydrphbic bases are stacked perpendicular t the axis f the helix n the inside. 1

2 The tw strands f DA are cmplementary and run in the ppsite directins, i.e., they are antiparallel. Althugh the bases are n the inside, they can be apprach thrugh tw deep spiral grves called the majr and minr grves. This mdel als accunted fr the fact that in the cmpsitin f DA the %A=%T and %G=%C. Stability f the DA Duplex DA duplex frmatin is favred by Base stacking (hydrphbic interactins). ydrgen bnd frmatin between base pairs. DA duplex frmatin is ppsed by Electrstatic repulsin f the backbne phsphates. Cnfrmatinal entrpy. ydrgen bnd frmatin with water. Duble elix Structures The duble helix f Watsn and Crick represents the structure f BDA. The-ray structure f DA basically cnfirmed the duble helix mdel, but shwed that the duble helix mdel was an versimplificatin. The mlecular structure shwed many lcal variatins and distrtins frm the idealized structure. It is imprtant t realize that DA secndary structure is nt rigid, but flexible, and depends n the exact nucletide sequence and culd be changed by interactins with prteins r ther mlecules. Anther helix, ADA is prduced under different cnditins. Recently, a left-handed ZDA has been described, but its physilgical significance is unknwn. A cmparisn f the three frms f the DA duble helix. The A frm is the helical cnfrmatin adpted by RA-RA (ARA) r RA-DA helices, because the 2 hydrxyl f ribse sterically inhibits frmatin f the B cnfrmatin. Mst DA in mst rganisms is dubled-stranded and mst dubledstranded DA is in the B frm. The mlecular structure suggests that the B frm is fund naturally because it, but nt the A frm, can accmmdate a spine f water mlecules (green) lying in the minr grve. The hydrgen bnds cntributed by the water give added stability t the B frm. In sme rganisms the DA is circular (n free 3 r 5 ends) and in thers linear. 2

3 The DA mlecules in eukarytes are immense The DA frm ne Drsphila melangaster (fruit fly) has a mlecular weight f 4 x g/ml (~6.5 x 10 7 base pairs) and wuld be 2 cm lng if fully extended (length = 0.34 nm x number f base pairs). The ttal DA frm all the cells in ne human wuld extend t sun and back 50 times! bviusly, these dimensins exceed the size f the rganisms and DA in the cells must be highly cndensed (see belw). RA - Secndary and Tertiary Structure There are three majr types f RA - transfer RA (tra); ribsmal RA (rra); and messenger RA (mra). ther minr frms f RA are imprtant fr splicing fr functins such as splicing and telmere synthesis. Transfer RA (tra) tras are small with nucletides - they functin in prtein synthesis. Single-stranded DA and RA have a tendency t adpt a randm cil structure. wever, such single-stranded mlecules cntain regins f self-cmplementary sequences where the mlecule can frm duble-stranded stems. tra cntains chemically mdified bases. Ribsmal RA (rra) The ribsme is a subcellular rganelle invlved in prtein synthesis. It cnsists f a large and a small subunit and rra is an integral part f the structure. rra is single stranded and is predicted t have a cmplex secndary structure, as depicted here fr 16 S rra frm E. cli. The ladder-like structures are hydrgen bnded base pairs. rra als cntains chemically mdified bases. 3

4 Recently (I.S. Gabashvili et al., Slutin Structure f the E. cli 70S Ribsme at 11.5 Å Reslutin, Cell, 100, (2000)) a mdel fr the ribsme has been cnstructed using cyr-electrn micrscpy. What yu see here are the prteinrra cmplexes f the large (50S) and small (30S) subunits. Messenger RA (mra) mra is the template fr prtein synthesis. It may assume an A-type duble helix r a single-stranded structure. mra des nt cntain chemically mdified bases. DA replicatin is semicnservative This means that the cpying mechanism invlves unwinding f the tw strands f a parental DA duplex, with each strand serving as the template fr the synthesis f a new strand, cmplementary t and wund abut the parental strand. At each base f the new strand, the cmplementary base t the parental strand is present and is held in psitin during plymerizatin by base pairing. Expressin f the genetic infrmatin in DA invlves transcriptin f the DA sequence int RA (messenger RA, mra), then translatin f the mra sequence int the prtein sequence. In frmatin f the mra, cmplementary base pairing between ribnucletides and the DA sequence is invlved. In the frmatin f prteins, each amin acid is specified by a sequence f the three bases (the cdn) t which a transfer RA, which carries the activated amin acid, binds. DA Denaturatin/Renaturatin Althugh the duble helix is relatively stable under physilgical cnditins, the lss f secndary structure, denaturatin, can be induced by: Enzymes- Fr example, RA plymerase, helicases. igh p, which leads t electrstatic repulsin f the negatively charged phsphate grups. These charges can be partially neutralized by ins r prteins 4

5 A change in the cncentratin f these "charge neutralizing" factrs can lead t chain separatin. Chemicals, such as frmamide, which decrease the base stacking energy f the duble helix. Anther factr cntrlling denaturatin is temperature due t the fact that the entrpy f the denatured state (randm cil) is higher than that f the helical state. The free energy change fr the helix randm cil transitin is G = - T. Fr the helix randm cil transitin electrstatic is negative and S cnfiguratinal is psitive - bth favrable. Therefre, in rder fr the duble helix t be stable under any cnditins there must be an unfavrable psitive D in the helix randm cil transitin that favrs the helical state. This psitive arises frm disruptin f the van der Waals interactins between the bases, which is the majr factr favring the helix. At physilgical temperature the magnitude f this psitive D must be greater than electrstatic - T S cnfiguratinal in rder fr the helix t be stable. wever, the sign f G will change frm + t - as the temperature increases due t the dminance f the entrpy term. In ther wrds the helix randm cil transitin will be favred at higher temperature. The extent f the denaturatin as temperature increases can be fllwed at 260 nm because the absrbance f the bases is less when stacked in the helix than in the randm cil state. The temperature f the transitin, T m, is quite sharp because the transitin is highly cperative - the DA is either in the helix state r the randm cil state. te that the curve fr refrmatin f the duble helix (annealing) is different (hysteresis) than the melting curve and that the annealing temperature T A is lwer than the T M. The sharpness f the melting curve suggests that melting is a highly cperative prcess (see belw). 5

6 The melting temperature T M, depends n bth the G+C cntent f the DA and the salt cncentratin f the slutin. Recall G-C base pairs are stabilized by 3 hydrgen bnds and A-T base pairs are stabilized by nly 2. The energy f the base stacking interactin between a G-C base pair and either a G-C r A-T base pair is strnger than the interactins with A-T base pairs. Thus a higher G-C cntent leads t higher duplex stability due t bth increased hydrgen bnding and base stacking interactins. igh inic strength shields the electrstatic repulsin f the phsphate grups in the backbne. Mdels fr melting and annealing f DA. Melting starts at the ends r internally at regins rich in AT base pairs. As the temperature increases the melted regins extend until the tw strands separate. Annealing is a bimlecular prcess, but there are cmpeting prcesses in which bth intrastrand and interstand base pairs can frm, ultimately leading t the stable duplex. 6

7 Cleavage f DA and RA Plymers Chemical Stability P - - P - + P P - - RA is susceptible t alkaline hydrlysis because f the presence f the free 2 ' - n ribse, as shwn in this diagram. DA is stable t alkaline hydrlysis, because it lacks the free 2 ' - n the ribse. ucleases ucleases are enzymes that hydrlyze the phsphdiester bnds in nucleic acids. Sme are specific fr DA, Dases, and thers fr RA, Rases and still thers shw n specificity. Exnucleases remve nucletides frm the ends, either frm the 5 ' - r 3 ' - ends. Endnucleases cleave internal phsphdiester bnds. Restrictin Enzymes Viruses that infect bacteria are called bacteriphages r phages. Phage cnsist f prtein and nucleic acid. Sme phages that grw well in ne strain f bacteria are ften unable t grw well in ther strains f bacteria. Phages d nt grw because their DA mlecules are cleaved and degraded by enzymes f the hst bacterial cell. Degrading DA destrys the ability f the phage t grw and is respnsible fr the pattern f grwth restrictin; hence the bacterial enzymes are called restrictin enzymes. Restrictin enzymes, f which nw mre than 1000 are knwn, are sequence-specific. Fr example, EcRI (frm E. cli) is specific fr the sequence GAATTC in duble stranded DA. ucleic Acids ybridize by Base Pairing A crucial prperty f the duble helix is the ability t separate the tw strands withut disrupting cvalent bnds. This makes it pssible fr the strands t separate and refrm under physilgical cnditins. The specificity f the prcess is determined by cmplementary base pairing. 7

8 ybridizatin refers t the frmatin f duble stranded nucleic acids. It is ften used t describe the frmatin f a DA-RA hybrid, as illustrated here. But annealing f DA is als hybridizatin. ybridizatin is als critical fr: The Plymerase Chain Reactin. Suthern Bltting, which invlved DA-DA hybridizatin. rthern Bltting, which invlves DA-RA hybridizatin. Assaying DA Micr Arrays. Tertiary Structure Because DA is such a lng mlecule, its tertiary structure is very cmplex. wever, we can discuss sme knwn structures. Superciling The DA f bacteria, mitchndria, plastids and sme viruses exists as a clsed circle. Circular DA can have a twisted r ciled appearance, as seen in these electrn micrscpe images (frm A. Krnberg DA Replicatin, pg. 29, W..Freeman, 1980). It is clear that superciling causes the DA t assume a mre cmpact structure. A clsed circle f DA can be frmed by jining the ends f linear DA tgether as shwn belw fr a 260 bp DA. 8

9 In bth the linear frm and the circular frm, the tw DA strands crss each ther 25 times. This is called the LIKIG UMBER, Lk, and is invariant n matter hw the circle is twisted. This circle is nt superciled and is called a relaxed circle. Superciling is induced if the DA is unwund befre (r after) the circle is made, as shwn belw. In frming this underwund circle we have changed the linking number frm 25 t 23. This DA circle is unstable because the DA duplex has been disrupted. The cmplete duplex can be refrmed if the circle then twists int a supercil as shwn belw. 9

10 We nw need tw additinal tplgical terms t describe ur superciled DA mlecule. ne is the TWIST, Tw, and the ther is the WRITE, Wr. Twist is the number f cmplete revlutins that ne strand makes abut the duplex axis. Fr B-DA the twist is the number f base pairs divided by 10.4 (the number f base pairs per turn f the B-DA duble helix). Fr this 260 bp DA, Tw = 25. Writhe is the number f times the helix axis crsses ver itself and is a measure f superciling. The helix f ur 260 bp mlecule makes tw right-handed crsses ver its self, s Wr = -2. Because Lk is cnstant fr any circle, fr every duble helical twist added, Tw, there must be an equal and ppsite supercil twist, - Wr. The usual value f Wr is negative, which means the supercil is right-handed. The tplgical state f a DA mlecule is described by the equatin: Lk = Tw + Wr. S fr ur 260 bp superciled mlecule: 23 = 25 + (-2). If ne f the three tplgical terms is changed, the ther terms must als change s that: Lk = Tw + Wr. Imprtance 1. Practical - gel separatin. Superciled DA mves faster n a gel (see belw). 2. Bilgical the tplgical state f the DA is critical fr its bilgical functin. 3. Packing f DA - Superciled DA is mre cmpact. Superciling is regulated enzymatically Tpismerase I relaxes supercils by increase the linking number in increments f 1 Gyrase induces superciling using ATP t decrease the linking number in increments f 2. This figure shws the effect f tpismerase I n the highly superciled DA f a virus. The enzyme intrduces a single strand break int the DA duplex, passes the ther strand thrugh the break and reseals the break. The net effect is t increase Lk and reduce Wr by +1. When Lk = Tw, the circle is relaxed. Agents that intercalate between the bases, such as ethidium brmide, unwind the DA duplex and cause superciling. 10

11 Chrmsmes Eukarytic DA is packaged int highly cndensed structures call chrmsmes. Belw are tw views f chrmsme rganizatin. uclesmes uclesme frmatin in eukarytic chrmatin invlves frming DA duplexes with a large writhe, which frm a trdial cil. The structure f a nuclesme cre particle has been determined. 11