Structure Determination

Structure Determination Reading Handout of Chapters 8 (intercalation) and 10 (sequencing) from

Blackburn & Gait Understand hydrolysis reaction used by nucleases (general mechanism) Know specificity of RNase T1, A, U2, and T2 Know specificity of S1 nuclease and DNase I Primary structure Describe Maxam-Gilbert sequencing for DNA or RNA If I give you a sequence you could describe how to sequence it DNA – use of DMS + pip, pip alone, hydrazine + pip RNA – use of DMA + aniline, DEPC + anil, hydrazine + anil Why do piperidine and aniline need to be used? Know the specificities of DMS for N7 of G and DEPC for N7 of A Understand use of RNases to sequence RNA (RNase T1, A, U2 and T2) Describe Sanger dideoxy sequencing Use of ddNTPs, fluorescent and radioactive tags Secondary structure

Understand how chemicals already stated can be used to determine base-pairing (single-stranded vs. double-stranded nucleotides)

Know the methods used: Radiolabel RNA/DNA, add chemical, induce cleavage, separate by

Electrophoresis Radiolabel RNA/DNA, add chemical, reverse transcription

Chemical probes to know: DMS – G ENU – phosphates DEPC – A CMCT – U/T (monitors Watson-Crick base pairing) OsO4 – T/U (faster reaction in single-stranded regions) Hydroxyl radical cleavage Advantage because of its small size Radicals generated by Fe(EDTA) + H2O2 + ascorbate Use of this technique for footprinting of proteins, RNA-RNA contacts,

cruciforms, hairpins, H-DNA Understand how intercalators are used with this technique

Tertiary structure Understand how all chemicals above can be used Know how the crosslinking agent psoralen helps determine 3˚ structure Understand how UV light can form crosslinks

Structure Determination

High resolution techniques - EM, NMR, X-ray

Biochemical techniques - 1. Use chemicals that react with DNA so that cleavage occurs at all bases (not sequence selective) so as to create a DNA/RNA cleavage ladder; alterations in uniform cleavage ladder can be used to footprint DNA/RNA

2. Basic strategy to probe DNA/RNA conformation with chemicals is to generate some type of conformation-related DNA/RNA modification that can be converted into a strand break which can then be mapped

Reagents react with one specific baseSpecificity with respect to: specific base specific base when it is in a unique conformation chemical not specific but method used to generate strand break is


Hydrolysis reaction:Used by nucleases (ribo- and deoxy-)Used by catalytic RNAs


Nucleases


Nucleases -restriction endonucleases

Sequence-specific

1. Compatible cohesive endsEcoRI restriction endonuclease

GAATTCCTTAAG

G AATTCCTTAA G

2. Blunt endsHaeIII restriction endonuclease

GGCCCCGG

GG CCCC GG

Structure DeterminationPrimary

Maxam-Gilbert chemical sequencing of DNAuses base-specific chemical reactions to break chain at A, G, C or T

G + A piperidine in acidC + T hydrazine and piperidine in baseG DMS and piperidine in baseC hydrazine, 1.5 M NaCl and piperidine in base

1. A base-specific rxn either cleaves aromatic ring of base or breaks glycosidic bond2. The first step destabilizes deoxyribose ring and allows attack by piperidine with subsequent ring cleavage3. Cleavage of phosphodiester chain4. Conversion of phosphodiester to phosphomonoester


Maxam-Gilbert chemical sequencing of DNA

G + A piperidine in acid



G DMS and piperidine in base



C + T hydrazine and piperidine in baseC hydrazine, 1.5 M NaCl and piperidine in base

5’-AATCGACTAGTACTAGTCTAGCTA-3’3’-TTAGCTGATCATGATCAGATCGAT-5’

Denature, 95 ˚C

5’-AATCGACTAGTACTAGTCTAGCTA-3’

3’-TTAGCTGATCATGATCAGATCGAT-5’+

Anneal radiolabeled primer

3’-TTAGCTGATCATGATCAGATCGAT-5’**5’-AATCGACTAGT

Extend with DNA polymerasedCTP, dATP, dGTP, dTTP

ddCTP

ddATP ddGTPddTTP

**5’-AATCGACTAGTddA

**5’-AATCGACTAGTAddC **5’-AATCGACTAGTACddT

**5’-AATCGACTAGTACTddA

**5’-AATCGACTAGTACTAddG

**5’-AATCGACTAGTACTAGddT**5’-AATCGACTAGTACTAGTddC

**5’-AATCGACTAGTACTAGTCddT

**5’-AATCGACTAGTACTAGTCTddA

**5’-AATCGACTAGTACTAGTCTAddG

**5’-AATCGACTAGTACTAGTCTAGddC

**5’-AATCGACTAGTACTAGTCTAGCddT

**5’-AATCGACTAGTACTAGTCTAGCTddA

DenatureGel electrophoresis

Structure Determination - PrimarySanger dideoxy sequencing of DNA

3’ 2’

Structure Determination - Primary

Sanger dideoxy sequencing of DNA

Structure Determination - PrimarySanger dideoxy sequencing of DNA


Determination of RNA sequence1. First convert RNA to DNA using reverse transcriptase

2. Peattie Chemical sequencing of RNA (similar to Maxam-Gilbert)uses base-specific chemical reactions to break chain at A, G, C or UUses aniline at pH 4.6 to cleave RNA at site of modification

G DMS and anilineU hydrazine and anilineA > G DEPC and anilineC > U hydrazine and 3 M NaCl and aniline

3. Ribonuclease sequencing RNases are base-specific, cut on the 3’ side of nucleotidelabel RNA at 5’ or 3’ end, add RNase, PAGE, also run OH- hydrolysis ladder


2. Peattie Chemical sequencing of RNA (similar to M-G)G DMS and anilineU hydrazine and anilineA > G DEPC and anilineC > U hydrazine and 3 M NaCl and aniline

Structure Determination - Secondary

Probe reactive sites with chemicals - if ds or ss only some sites can be modified

For RNA and DNA covalent rxns can reveal which bases are (i) single-stranded (unbonded and usually most reactive), (ii) which are in base-pairs (hydrogen bonded) and (iii) which are maybe involved in 3˚ interactions

Precise locations of binding of proteins & drug molecules can be determined by differences in reactivity of bound and naked DNA

Experiments:Radiolabel DNA or RNAAt 5’-end with [-32P] ATP and polynucleotide kinaseAt 3’-end with RNA ligase and radioactive pNp

Chemical reactivity determined by:1. Chemical cleavage of an end-labeled chain at site of modification

refer to M-G seq and use of aniline (RNA) and piperidine (DNA)2. Chain termination by reverse transcriptase at this site RT makes a complementary copy of RNA or DNA single strands,

but it stops or pauses at modified bases or at the end of the strand where cleavage occurred. Length of copy identifies location of modified base (seq rxns done in parallel to provide exact position)


Probe reactive sites in DNA/RNA with chemicals


Probe reactive sites with chemicalsAll nitrogens and oxygens in base, sugar & phosphate can be alkylated

Rxn

s w

ith

A a

nd C

<<

in d

sDN

A

Structure Determination - SecondaryProbe reactive sites with chemicals - all these monitor W-C base pairing abilityGood probes of duplex formation

Structure Determination - SecondaryProbe reactive sites with chemicals - react faster in ss regions

Structure Determination - SecondaryRNA - use all reagents in Table 3-2

Base specific nucleases (Table 3-1) are all more reactive on ssRNA than on dsRNA

S1 hydrolyzes ssRNA or ssDNA but it is inhibited by ds structure Ex: in tRNA only anticodon loop is cutRNase V1 is specific for dsRNA and stacked ssRNA

LIMITS of Nucleases - size of enzymes limit accessibility to site

OH- cleavage nonspecific cleavage of backbone


DNA - mostly duplex but do get ssDNA at cruciforms, triplex, junctionsCan also use chemical reagents to determine where proteins bind - FOOTPRINT

Cruciforms - ss loops hyperactive to ss-selective reagents bromoacetaldehyde, kethoxal, OsO4, bisulfite

Triplex - induced in polypurine rich regions by (-) supercoiling and low pH, in H-DNA ss released and becomes hyperactive to ss-selective reagents, also G in triplex is protected from alkylation at N7 by DMS

Nucleases - S1 hydrolyzes ssDNA/RNA

FeII(EDTA) is an effective cleaving agent because hydroxyl radicals are oxidative agents that form when ferrous ions are in the presence of O2 (H2O2) and a reducing agent (ascorbate)•OH cleaves the sugar-phosphate backbone of DNAFeII(EDTA) complexes have been applied to DNA and to probing the solvent accessibility of RNA



DNA

Fe(EDTA)2- + H2O2 + ascorbate

•OH •OH •OH•OH

Fe2+

Fe3+

H2O2

•OHOH-

OBase

O

P

O

O O-

O

P

O

O O-

•OH

O-

P

O

O O-

O

P

O-

O O-Base falls off


DNAMethidiumpropyl-EDTA + Fe (II) + O2

This reagent binds via intercalation, with little sequence discrimination and delivers oxidizing agent to the DNA


NAIM - Nucleotide Analog Interference Mapping

• Allows simultaneous, yet individual, identification of important functional groups within a large RNA

• Nucleotide analogs are randomly incorporated into RNA during in vitro transcription

• Selective assay for RNA function to separate active from inactive ribozymes

• Analogs have a chemical tag - phosphorothioate linkage - to identify site of incorporation


NAIM - Nucleotide Analog Interference Mapping

Secondary versus tertiary structure

• Secondary structure can be drawn in 2D without any crossing lines

• Tertiary interactions cross the lines of the secondary structure (chord crossing)

Structure Determination - Tertiary

1. use all chemicals/enzymes used for secondary structure because tertiary contacts can protect regions from chemical modification or nuclease cleavage

2. Chemical crosslinking - using psoralen and/or UV lightreagents have 2 reactive groups that can be used to covalently link nucleotides that are far apart in sequence; determine location by EM or partial hydrolysis

Combine crosslinking data with chemical probing and phylogeny to get a precise 3-dimensional structure


1. Intercalation of psoralen (intercalators are planar and stack as bases)2. Psoralen absorbs a photon ( = 300-400 nm)3. C=C on psoralen reacts by cycloaddition to C5=C6 of pyrimidine4. Psoralen can then form a second crosslink to another pyrimidine

T, U >> C

Chemical crosslinking - using psoralenUsed with many nucleic acids, such as 16S rRNA and viral DNA


Chemical crosslinking - using UV light

Use 200-300 nm light to cause crosslinks in DNAFormation of pyrimidine dimers linked by a cyclobutyl ring across C5=C6Advantage because no chemical agent has to be added to solution


Intercalation - planar aromatic molecules binding to DNA


Intercalation - separation of base pairs with a lengthening of double helix and a decrease in helical twist (unwinding)


Intercalator - ditercalinium is a bisintercalator (rigid chain)

Used as an anticancer drug because it induces DNA repair in many spots on the cell’s DNA (nonspecific, noncovalent binding) which eventually causes the cell to die because of extensive repair-induced DNA degradation

23S rRNA (then & now)

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Structure Determination