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