01 may 2008 nucleic acid chemistry andy howard introductory biochemistry 1 may 2008

01 May 2008Nucleic Acid Chemistry

Nucleic Acid Chemistry

Andy HowardIntroductory Biochemistry

1 May 2008

01 May 2008Nucleic Acid Chemistry p.2 of 43

What we’ll discuss

RNA (concluded) Chromatin

Packaging of DNA Nucleosomes Histones Higher levels Bacterial

packaging

Nucleases Alkaline hydrolysis RNAses Restriction

Endonucleases Applications of

restriction endos


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 by DS?

mRNA 3 25 50-104 no protein template

tRNA 15 21 55-90 yes aa activation

rRNA 80 50 102-104 no transl. catalysis &

scaffolding

sRNA 2 4 30-103 ? various


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!


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


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


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


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


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


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)


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


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


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


Chromatin Discovered long before we

understood molecular biology

Seen to be banded objects in nuclei of stained eukaryotic cells

In resting cell it exists as long slender threads, 30 nm diameter From answers.com


Squishing the DNA If the double helix were fully extended,

the largest human chromosome (2.4*108bp) would be 2.4*108 *0.33nm ~ 0.8*108nm=80 mm;

much bigger than the cell! So we have to coil it up a lot to make it fit. Longest chromosome is 10µm long So the packing ratio is 80mm/10µm =

8000


Nucleosomes DNA-protein complexes that

hold together the DNA in coiled forms at the second- and third- levels of organization(first is helicity itself)

The proteins involved are histones Proteins rich in basic aa’s (R,K) These interact closely with DNA to

facilitate appropriate coiling

Nucleosomecore particlePDB 1KX5143 bp +108 kDa heterooctamerXenopus


Histones Characterized as H1, H2A, H2B, H3, H4 H1 involved in higher level of

organization; others in nucleosome itself All are small, K&R-rich proteins Highly conserved


Categories of histones

Type MW,kDa #aa’s #basic #acidic

H1 21 213 65 10

H2A 14 129 30 9

H2B 13.8 125 31 10

H3 15.3 135 33 11

H4 11.3 102 27 7


Unfolded chromatin Treat chromatin with low ionic strength;

that disrupts higher level interactions so the individual nucleosomes are strung out relative to one another like beads on a string

Image courtesy U. Maine


Histone deactivation Histones interact with DNA via

+charges on lys and arg residues. If we neutralize those charges by

acetylation, the histones don’t bind as tightly to the DNA

Carefully-timed enzymatic control of histone acylation is a crucial element in DNA organization

NH3+

HN

O

O-

acylated lysineO


Histone acetylation

Active histone + Acetyl CoA inactive (acetylated) histone + CoASH

Without the positive charges, the affinity for DNA goes down

CoASH

Histone H1PDB 1GHC8.3 kDa monomerChicken Histone

acetyltransferasePDB 1QSO

66 kDatetramer

yeast


Histone deacetylation

Type III deacetylases usea non-trivial reaction:Prot-lys-NAc + NAD+ Prot-lys-NH3

+ + nicotinamide +2’-O-acetyl-ADP-ribose

Part of the NAD salvage pathway Histone/protein deacetylase +

histone H4 active peptidePDB 1SZD; 34 kDa “heterodimer”yeast


Nucleosome structure

Core octamer is two molecules each of H2A, H2B, H3, H4

Typically wraps around~200bp of DNA

DNA betweennucleosomes is ~54 bp long

H1 binds to linker and to core particle; but in beads-on-a-string structure, it’s often absent


How much does this coil up?

200 bp extended would be about 50nm The width of the core-particle disk is 5nm So this is a tenfold reduction Nucleosomal organization corresponds to

negative supercoiling … so DNA ends up supercoiled when we

take away the histones


Next level of organization

H1 interacts with DNA along linker region

Individual histones spiral along to form 30 nm fiber

See fig.19.25

Courtesy answers.com

Courtesy Johns Hopkins Univ


Even higher… The 30nm fibers are attached to

an RNA-protein scaffold that holds the 30nm fibers in large loops

Typical chromosome has ~200 loops

Loops are attached to scaffold at their base

Ends can rotate so it can be supercoiled


What about prokaryotes? No actual histones Histone-like proteins involved Bacterial DNA attached to

scaffold in large loops (~100kb) This makes a nucleoid


How many loops in bacteria? Typical bacterial genome (E.coli) has

3000 open reading frames ~ 3000 genes.

Assume 500 amino acids per protein = 1500 bases per gene (ignores transcriptional elements)

Then genome is 1500 bp/gene * 3000 genes = 4.5*106 base-pairs

That’s (4.5*106 bp)/(1*105 bp/loop) = 45 loops


Nucleases Enzymes that hydrolyze

phosphodiester bonds in nucleic acids

Can clip on the 3’ end or the 5’ end of the phosphorus

Can operate on DNA or RNA DNA tends to be more

resistant to degradation

P

O

O--O

O

OH

O

NO

HN

O

P

O

O-O

O

HO

OH

O

NN

NH2

N

N


Alkaline hydrolysis RNA can be readily hydrolyzed

nonenzymatically, particularly at high pH DNA considerably less so RNA will be completely degraded at pH

13 (0.1N NaOH) in hours;DNA untouched

This will still happen at lower pH, but much more slowly


Mechanism for alkaline hydrolysis of RNA (fig. 19.28)

Cyclic phosphate intermediate stabilizes cleavage product


Results of creating cyclic phosphate 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-

O-

H

O

H

H

O

H

H


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!


Enzymatic hydrolysis of RNA

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


Restriction endonucleases

These are sequence-specific enzymes that cleave phosphodiester bonds in DNA

Found in bacteria, which use them to cleave foreign DNA

EcoRI with DNAPDB 1ERI61 kDa dimer + 13 bpE.coli (obviously)


The biology problem How does the bacterium mark its own DNA so

that it does replicate its own DNA but not the foreign DNA?

Answer: by methylating specific bases in its DNA prior to replication

Unmethylated DNA from foreign source gets cleaved by restriction endonuclease

Only the methylated DNA survives to be replicated

Most methylations are of A & G,but sometimes C gets it too


How it works When an unmethylated specific

sequence appears in the DNA, the enzyme cleaves it

When the corresponding methylated sequence appears, it doesn’t get cleaved and remains available for replication

The restriction endonucleases only bind to palindromic sequences


Palindromic DNA Sequences that read the same on one

strand from left to right as they do on the opposite strand reading right to left

Example, found in EcoRI recognition sequence:5’-GAATTC-3’3’-CTTAAG-5’

Most DNA isn’t palindromic, but palindromic sequences are common enough that we can frequently find them


Nomenclature for restriction endonucleases (table 19.4) Name has three pieces:

3- or 4-character designation for organism, e.g. Eco (E.coli), Kpn (Klebsiella pneoumoniae), Bam (Bacillus amyloliquefaciens)

(optional) one-character designation for strain (R, H) (e.g. R is strain R of E.coli)

Roman-numeral characters for enzyme

Thus :EcoRI, BamH1 Simpler: XbaI


Generalizations about restriction endonucleases Each binds a specific 4-7 base-pair

sequence Always recognize palindromic sequences;

often local dimer within enzyme lines up on the two identical strands of DNA

Cleavage site can be anywhere within the sequence

Methylation site typically not on the cleaved base


Common lab endonucleasesNuclease Source Sequence CutApaI Acetobacter 5’GGGCCC 5’-GGGCCC C-3’ pasteurianus 3’CCCGGG 3’-C CCGGG-5’BamHI Bacillus amilo5’GGATCC 5’-G GATCC-3’

-liquifaciens3’CCTAGG 3’-CCTAG G-5’

EcoRI Escherichia 5’GAA*TTC 5’-G AATTC-3’

coli 3’CTT*AAG 3’-CTTAA G-5’

EcoRII E.coli 5’CC*WGG 5’- CCWGG-3’3’GG*WCC 5’-GGWCC -5’

HinDIII Haemophilus 5’A*AGCTT 5’-A AGCTT-3’ influenzae 3’T*TCGAA 3’-TTCGA A-5’

HpaII Haemophilus 5’CCGG 5’-C CGG-3’ parainflu. 3’GGCC 3’-GGC C-5’

Cf. table 19.4!


More endonucleasesNuclease Source Sequence CutKpnI Klebsiella 5’GGTACC 5’-GGTAC C-3’

pneumoniae 3’CCATGG 3’-C CATGG-5’NotI Nocardia 5’GCGGCCGC 5’-GC GGCCGC-3’

otitidis 3’CGCCGGCG 3’-CGCCGG CG-5’PstI Providencia 5’CTGCAG 5’-CTGCA G-3’

stuartii 164 3’GACGTC 3’-G ACGTC-5’SmaI Serratia 5’CCCGGG 5’-CCC GGG-3’

marescens 3’GGGCCC 3’-GGG CCC-5’XbaI Xanthomonas5’TCTAGA 5’-T CTAGA-3’

badrii 3’AGATCT 3’-AGATC T-5’XhoI Xanthomonas5’CTCGAG 5’-C TCGAG-3’

holcicola 3’GAGCTC 3’-GAGCT C-5’TaqI Thermus 5’TCGA 5’-T CGA-3’

aquaticus 3’AGCT 3’-AGC T-5’


Applications Cleaving DNA at the restriction sites

Building cleavable constructs in plasmids Recombinant DNA depends on identifying

restriction sites and cleaving them

Identifying mutations in a population That allows studies of genetic drift DNA fingerprinting in forensics Can be combined with PCR so the starting

DNA sample can be very small

01 may 2008 nucleic acid chemistry andy howard introductory biochemistry 1 may 2008

Documents

bases unmodified mrna

number of bases

bases long15

bases moleculeoften

unwanted bases

nucleic acid chemistrywhat

processing of mrna

final mrna