10/07/2010Biochemistry:Nucleic Acids II Nucleic Acids: DNA, RNA and chemistry Andy Howard...

10/07/2010Biochemistry:Nucleic Acids II

Nucleic Acids:DNA, RNA and chemistry

Andy HowardIntroductory Biochemistry

7 October 2010

10/07/2010 Biochemistry:Nucleic Acids II

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DNA & RNA structure & function DNA and RNA are dynamic molecules, but understanding their structural realities helps us understand how they work


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What we’ll discuss DNA structure

Characterizations B, A, and Z-DNA Dynamics Function

RNA:structure & types mRNA tRNA rRNA Small RNAs

DNA & RNA Hydrolysis alkaline RNA, DNA nucleases


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DNA secondary structures If double-stranded DNA were simply a straight-legged ladder: Base pairs would be 0.6 nm apart Watson-Crick base-pairs have very uniform dimensions because the H-bonds are fixed lengths

But water could get to the apolar bases So, in fact, the ladder gets twisted into a helix.

The most common helix is B-DNA, but there are others. B-DNA’s properties include: Sugar-sugar distance is still 0.6 nm Helix repeats itself every 3.4 nm, i.e. 10 bp


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Properties of B-DNA

Spacing between base-pairs along helix axis = 0.34 nm

10 base-pairs per full turn

So: 3.4 nm per full turn is pitch length

Major and minor grooves, as discussed earlier

Base-pair plane is almost perpendicular to helix axis

From Molecular Biology web-book


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Major groove in B-DNA

H-bond between adenine NH2 and thymine ring C=O

H-bond between cytosine amine and guanine ring C=O

Wide, not very deep


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Minor groove in B-DNA

H-bond between adenine ring N and thymine ring NH

H-bond between guanine amine and cytosine ring C=O

Narrow but deepFrom Berg et al.,Biochemistry


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Cartoon of AT pair in B-DNA


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Cartoon of CG pair in B-DNA


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What holds duplex B-DNA together?

H-bonds (but just barely) Electrostatics: Mg2+ –PO4

-2

van der Waals interactions - interactions in bases Solvent exclusion

Recognize role of grooves in defining DNA-protein interactions


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Helical twist (fig. 11.9a) Rotation about the backbone axis

Successive base-pairs rotated with respect to each other by ~ 32º


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Propeller twist

Improves overlap of hydrophobic surfaces

Makes it harder for water to contact the less hydrophilic parts of the molecule


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A-DNA (figs. 11.10) In low humidity this forms

naturally Not likely in cellular duplex DNA,but it does form in duplex RNA & DNA-RNA hybrids because the2’-OH gets in the way of B-RNA

Broader 2.46 nm per full turn 11 bp to complete a turn

Base-pairs are notperpendicular to helix axis:tilted 19º from perpendicular


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Z-DNA (figs.11.10)

Forms in alternating Py-Pu sequences and occasionally in PyPuPuPyPyPu, especially if C’s are methylated

Left-handed helix rather than right

Bases zigzag across the groove


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Getting from B to Z Can be accomplished without breaking bonds

… even though purines have their glycosidic bonds flipped (anti -> syn) and the pyrimidines are flipped altogether!


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Summaries of A, B, Z DNA


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DNA is dynamic Don’t think of these diagrams as static

The H-bonds stretch and the torsions allow some rotations, so the ropes can form roughly spherical shapes when not constrained by histones

Shape is sequence-dependent, which influences protein-DNA interactions


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What does DNA do? Serve as the storehouse and the propagator of genetic information:That means that it’s made up of genes Some code for mRNAs that code for protein Others code for other types of RNA Genes contain non-coding segments (introns)

But it also contains stretches that are not parts of genes at all and are serving controlling or structural roles

Avoid the term junk DNA!


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Ribonucleic acid We’re done with DNA for the moment. Let’s discuss RNA. RNA is generally, but not always, single-stranded

The regions where localized base-pairing occurs (local double-stranded regions) often are of functional significance


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RNA physics & chemistry

RNA molecules vary widely in size, from a few bases in length up to 10000s of bases

There are several types of RNA found in cellsType %%turn- Size, Partly Role

RNA over bases DS?mRNA 3 25 50-104 no protein

templatetRNA 15 21 55-90 yes aa activationrRNA 80 50 102-104 no transl.

catalysis & scaffolding

sRNA 2 4 15-103 ? various


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Messenger RNA mRNA: transcription vehicleDNA 5’-dAdCdCdGdTdAdTdG-3’RNA 3’- U G G C A U A C-5’

typical protein is ~500 amino acids;3 mRNA bases/aa: 1500 bases (after splicing)

Additional noncoding regions (see later) brings it up to ~4000 bases = 4000*300Da/base=1,200,000 Da

Only about 3% of cellular RNA but instable!


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Relative quantities

Note that we said there wasn’t much mRNA around at any given moment

The amount synthesized is much greater because it has a much shorter lifetime than the others

Ribonucleases act more avidly on it We need a mechanism for eliminating it because the cell wants to control concentrations of specific proteins


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mRNA processing in Eukaryotes

# bases (unmodified mRNA) = # base-pairs of DNA in the gene…because that’s how transcription works

BUT the number of bases in the unmodified mRNA > # bases in the final mRNA that actually codes for a protein

SO there needs to be a process for getting rid of the unwanted bases in the mRNA: that’s what splicing is!

Genomic DNA

Unmodified mRNA produced therefrom


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Splicing: quick summary

Typically the initial eukaryotic message contains roughly twice as many bases as the final processed message

Spliceosome is the nuclear machine (snRNAs + protein) in which the introns are removed and the exons are spliced together

Genomic DNA

Unmodified mRNA produced therefrom

exon intron exon exonintron intron

exon exon exonsplicing

translation

transcription

(Mature transcript)


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Heterogeneity via spliceosomal flexibility Specific RNA sequences in the initial mRNA signal where to start and stop each intron, but with some flexibility

That flexibility enables a single gene to code for multiple mature RNAs and therefore multiple proteins


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Transfer RNA tRNA: tool for engineering protein synthesis at the ribosome

Each type of amino acid has its own tRNA, responsible for positioning the correct aa into the growing protein

Roughly T-shaped or Y-shaped molecules; generally 55-90 bases long

15% of cellular RNA

Phe tRNAPDB 1EVV76 basesyeast


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Secondary and Tertiary Structure of tRNA Extensive H-bonding creates four double helical domains, three capped by loops, one by a stem

Only one tRNA structure (alone) is known

Phenylalanine tRNA is "L-shaped" Many non-canonical bases found in tRNA


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tRNA structure: overview


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Amino acid linkage to acceptor stem

Amino acids are linked to the 3'-OH end of tRNA molecules by an ester bond formed between the carboxyl group of the amino acid and the 3'-OH of the terminal ribose of the tRNA.


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Yeast phe-tRNA Note nonstandard bases and cloverleaf structure


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Ribosomal RNA

rRNA: catalyic and scaffolding functions within the ribosome

Responsible for ligation of new amino acid (carried by tRNA) onto growing protein chain

Can be large: mostly 500-3000 bases

a few are smaller (150 bases) Very abundant: 80% of cellular RNA

Relatively slow turnover

23S rRNAPDB 1FFZ602 basesHaloarcula marismortui


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Small RNA sRNA: few bases / molecule often found in nucleus; thus

it’s often called small nuclear RNA, snRNA

Involved in various functions, including processing of mRNA in the spliceosome

Some are catalytic Typically 20-1000 bases Not terribly plentiful: ~2 %

of total RNA

Protein Prp31complexed to U4 snRNAPDB 2OZB33 bases + 85kDa heterotetramerHuman


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iClicker quiz 1. Shown is the lactim form of which nucleic acid base? Uracil Guanine Adenine Thymine None of the above

HN

O N OH

lactim


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iClicker quiz #2 Suppose someone reports that he has characterized the genomic DNA of an organism as having 29% A and 22% T. How would you respond?

(a) That’s a reasonable result (b) This result is unlikely because [A] ~ [T] in duplex DNA

(c) That’s plausible if it’s a bacterium, but not if it’s a eukaryote

(d) none of the above


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Unusual bases in RNA mRNA, sRNA mostly ACGU

rRNA, tRNA have some odd ones


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Other small RNAs 21-28 nucleotides

Target RNA or DNA through complementary base-pairing

Several types, based on function: Small interfering RNAs (q.v.) microRNA: control developmental timing

Small nucleolar RNA: catalysts that (among other things) create the oddball bases QuickTime™ and a

TIFF (Uncompressed) decompressorare needed to see this picture.snoRNA77

courtesy Wikipedia


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siRNAs and gene silencing Small interfering RNAs block

specific protein production by base-pairing to complementary seqs of mRNA to form dsRNA

DS regions get degraded & removed

This is a form of gene silencing or RNA interference

RNAi also changes chromatin structure and has long-range influences on expression

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Viral p19 protein complexed to human 19-base siRNAPDB 1R9F1.95Å17kDa protein


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Do the differences between RNA and DNA matter? Yes!

DNA has deoxythymidine, RNA has uridine: cytidine spontaneously degrades to uridine dC spontaneously degrades to dU

The only dU found in DNA is there because of degradation: dT goes with dA

So when a cell finds dU in its DNA, it knows it should replace it with dC or else synthesize dG opposite the dU instead of dA


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Ribose vs. deoxyribose Presence of -OH on 2’ position makes the 3’ position in RNA more susceptible to nonenzymatic cleavage than the 3’ in DNA

The ribose vs. deoxyribose distinction also influences enzymatic degradation of nucleic acids

I can carry DNA in my shirt pocket, but not RNA


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Backbone hydrolysis of nucleic acids in base(fig. 10.29)

Nonenzymatic hydrolysis in base occurs with RNA but not DNA, as just mentioned

Reason: in base, RNA can form a specific 5-membered cyclic structure involving both 3’ and 2’ oxygens

When this reopens, the backbone is cleaved and you’re left with a mixture of 2’- and 3’-NMPs


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Why alkaline hydrolysis works Cyclic phosphate intermediate stabilizes cleavage product


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The cyclic intermediate

Hydroxyl or water can attack five-membered P-containing ring on either side and leave the –OP on 2’ or on 3’.

P

O

O-

O-

O

OO

ON

OHN

O

P

O

O-


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Consequences So RNA is considerably less stable compared to DNA, owing to the formation of this cyclic phosphate intermediate

DNA can’t form this because it doesn’t have a 2’ hydroxyl

In fact, deoxyribose has no free hydroxyls!


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Enzymatic cleavage of oligo- and polynucleotides Enzymes are phosphodiesterases Could happen on either side of the P 3’ cleavage is a-site; 5’ is b-site. Endonucleases cleave somewhere on the interior of an oligo- or polynucleotide

Exonucleases cleave off the terminal nucleotide


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An a-specific exonuclease


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A b-specific exonuclease


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Specificity in nucleases

Some cleave only RNA, others only DNA, some both

Often a preference for a specific base or even a particular 4-8 nucleotide sequence (restriction endonucleases)

These can be used as lab tools, but they evolved for internal reasons


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Enzymatic RNA hydrolysis

Ribonucleases operate through a similar 5-membered ring intermediate: see fig. 19.29 for bovine RNAse A: His-119 donates proton to 3’-OP His-12 accepts proton from 2’-OH

Cyclic intermediate forms with cleavage below the phosphate

Ring collapses, His-12 returns proton to 2’-OH, bases restored

PDB 1KF813.6 kDa monomerbovine


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Variety of nucleases

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