Nucleic Acids: RNA and chemistry

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10/13/2009 Biochemistry:Nucleic Acids II Nucleic Acids: RNA and chemistry Andy Howard Introductory Biochemistry 13 October 2009

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Nucleic Acids: RNA and chemistry. Andy Howard Introductory Biochemistry 13 October 2009. RNA: structure & types mRNA tRNA rRNA Small RNAs. DNA & RNA Hydrolysis alkaline RNA, DNA nucleases Restriction enzymes DNA & RNA dynamics and density measurements. What we’ll discuss. - PowerPoint PPT Presentation

Transcript of Nucleic Acids: RNA and chemistry

Page 1: Nucleic Acids: RNA and chemistry

10/13/2009Biochemistry:Nucleic Acids II

Nucleic Acids:RNA and chemistry

Andy HowardIntroductory Biochemistry

13 October 2009

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What we’ll discuss RNA: structure &

types mRNA tRNA rRNA Small RNAs

DNA & RNA Hydrolysis alkaline RNA, DNA nucleases

Restriction enzymes

DNA & RNA dynamics and density measurements

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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 30-103 ? 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 base pairs 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 ala-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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Ribosomal composition (fig.10.22) Bacterial ribosome

30S subunit: 16S RNA + 21 proteins 50S subunit:23S RNA + 5S RNA + 31 proteins

Eukaryotic ribosome 40S subunit: 18S RNA + 33 proteins 60S subunit:(28S+5.85S) RNA, 5S RNA + 49 proteins

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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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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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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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How many varieties of each class? mRNA: thousands(one per protein transcript)

tRNA: one per codon plus a few more

rRNA: a few per organism—see rRNA slide

sRNA: dozens (?)

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Unusual bases in RNA mRNA, sRNA mostly ACGU

rRNA, tRNA have some odd ones

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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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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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Restriction endonucleases

Evolve in bacteria as antiviral tools “Restriction” because they restrict the incorporation of foreign DNA into the bacterial chromosome

Recognize and bind to specific palindromic DNA sequences and cleave them

Self-cleavage avoided by methylation Types I, II, III: II is most important I and III have inherent methylase activity; II has methylase activity in an attendant enzyme

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What do we mean by palindromic?

In ordinary language, it means a phrase that reads the same forward and back: Madam, I’m Adam. (Genesis 3:20) Eve, man, am Eve. Sex at noon taxes. Able was I ere I saw Elba. (Napoleon) A man, a plan, a canal: Panama! (T. Roosevelt)

With DNA it means the double-stranded sequence is identical on both strands

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Quirky math question to ponder Numbers can be palindromic:484, 1331, 727, 595…

Some numbers that are palindromic have squares that are palindromic…222 = 484, 1212 = 14641, . . .

Question: if a number is perfect square and a palindrome, is its square root a palindrome? (answer will be given orally)

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Palindromic DNA G-A-A-T-T-C Single strand isn’t symmetric: but the combination with the complementary strand is:

G-A-A-T-T-CC-T-T-A-A-G

These kinds of sequences are the recognition sites for restriction endonucleases. This particular hexanucleotide is the recognition sequence for EcoRI.

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Cleavage by restriction endonucleases

Breaks can be cohesive (if they’re off-center within the sequence) or

non-cohesive (blunt) (if they’re at the center) EcoRI leaves staggered 5’-termini: cleaves between initial G and A

PstI cleaves CTGCAG between A and G, so it leaves staggered 3’-termini

BalI cleaves TGGCCA in the middle: blunt!

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iClicker question 3:

3. Which of the following is a potential restriction site? (a) ACTTCA (b) AGCGCT (c) TGGCCT (d) AACCGG (e) none of the above.

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Example for EcoRI 5’-N-N-N-N-G-A-A-T-T-C-N-N-N-N-3’3’-N-N-N-N-C-T-T-A-A-G-N-N-N-N-5’

Cleaves G-A on top, A-G on bottom: 5’-N-N-N-N-GA-A-T-T-C-N-N-N-N-3’3’-N-N-N-N-C-T-T-A-AG-N-N-N-N-5’

Protruding 5’ ends:5’-N-N-N-N-G A-A-T-T-C-N-N-N-N-3’3’-N-N-N-N-C-T-T-A-A G-N-N-N-N-5’

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How often?

4 types of bases So a recognition site that is 4 bases long will occur once every 44 = 256 bases on either strand, on average

6-base site: every 46= 4096 bases, which is roughly one gene’s worth

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EcoRI structure

Dimeric structure enables recognition of palindromic sequence

sandwich in each monomer

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

EcoRI pre-recognition complexPDB 1CL857 kDa dimer + DNA

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Methylases A typical bacterium protects its own DNA against cleavage by its restriction endonucleases by methylating a base in the restriction site

Methylating agent is generally S-adenosylmethionine

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

HhaI methyltransferasePDB 1SVU2.66Å; 72 kDa dimer

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Structure courtesy steve.gb.com

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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

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How this 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