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Nucleic Acids: RNA and chemistry

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Title: Nucleic Acids: RNA and chemistry Author: Andrew Howard User Last modified by: Andrew Howard User Created Date: 10/12/2009 11:58:35 PM Document presentation format – PowerPoint PPT presentation

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Title: Nucleic Acids: RNA and chemistry


1
Nucleic Acids RNA and chemistry
  • Andy Howard Introductory Biochemistry 13 October
    2009

2
What well discuss
  • DNA RNA Hydrolysis
  • alkaline
  • RNA, DNA nucleases
  • Restriction enzymes
  • DNA RNA dynamics and density measurements
  • RNA structure types
  • mRNA
  • tRNA
  • rRNA
  • Small RNAs

3
Ribonucleic acid
  • Were done with DNA for the moment.
  • Lets 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

4
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 cells
  • Type turn- Size, Partly Role
  • RNA over bases 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

5
Messenger RNA
  • mRNA transcription vehicle DNA
    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 4000300Da/base1,200,000
    Da
  • Only about 3 of cellular RNA but instable!

6
Relative quantities
  • Note that we said there wasnt 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

7
mRNA processing in Eukaryotes
Genomic DNA
Unmodified mRNA produced therefrom
  • bases (unmodified mRNA) base-pairs of DNA
    in the gene because thats how transcription
    works
  • BUT the number of bases in the unmodified mRNA gt
    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 thats what
    splicing is!

8
Splicing quick summary
Genomic DNA
transcription
Unmodified mRNA produced therefrom
exon
intron
exon
exon
intron
intron
splicing
exon
exon
exon
translation
(Mature transcript)
  • 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

9
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

10
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 tRNA PDB 1EVV 76 bases yeast
11
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

12
tRNA structure overview
13
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.

14
Yeast ala-tRNA
  • Note nonstandard bases and cloverleaf structure

15
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 rRNA PDB 1FFZ 602 bases Haloarcula marismortui
16
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 (28S5.85S) RNA, 5S RNA 49 proteins

17
Small RNA
  • sRNA few bases / molecule
  • often found in nucleus thus its 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 Prp31 complexed to U4 snRNA PDB 2OZB 33
bases 85kDa heterotetramer Human
18
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

Viral p19 protein complexed to human 19-base
siRNA PDB 1R9F 1.95Å 17kDa protein
19
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

snoRNA77 courtesy Wikipedia
20
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 (?)

21
Unusual bases in RNA
  • mRNA, sRNA mostly ACGU
  • rRNA, tRNA have some odd ones

22
iClicker quiz
  • 1. Shown is the lactim form of which nucleic acid
    base?
  • Uracil
  • Guanine
  • Adenine
  • Thymine
  • None of the above

23
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) Thats a reasonable result
  • (b) This result is unlikely because A T in
    duplex DNA
  • (c) Thats plausible if its a bacterium, but not
    if its a eukaryote
  • (d) none of the above

24
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

25
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

26
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
    youre left with a mixture of 2- and 3-NMPs

27
Why alkaline hydrolysis works
  • Cyclic phosphate intermediate stabilizes cleavage
    product

28
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.

29
Consequences
  • So RNA is considerably less stable compared to
    DNA, owing to the formation of this cyclic
    phosphate intermediate
  • DNA cant form this because it doesnt have a 2
    hydroxyl
  • In fact, deoxyribose has no free hydroxyls!

30
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

31
An a-specific exonuclease
32
A b-specific exonuclease
33
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

34
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 1KF8 13.6 kDa monomer bovine
35
Variety of nucleases
36
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

37
What do we mean by palindromic?
  • In ordinary language, it means a phrase that
    reads the same forward and back
  • Madam, Im Adam. (Genesis 320)
  • 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

38
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)

39
Palindromic DNA
  • G-A-A-T-T-C
  • Single strand isnt symmetric but the
    combination with the complementary strand is
  • G-A-A-T-T-C C-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.

40
Cleavage by restriction endonucleases
  • Breaks can be
  • cohesive (if theyre off-center within the
    sequence) or
  • non-cohesive (blunt) (if theyre 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!

41
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.

42
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-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
  • 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

43
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 genes worth

44
EcoRI structure
  • Dimeric structure enables recognition of
    palindromic sequence
  • ??? sandwich in each monomer

EcoRI pre-recognition complex PDB 1CL8 57 kDa
dimer DNA
45
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-adenosylmethionin
    e

HhaI methyltransferase PDB 1SVU 2.66Å 72 kDa
dimer
Structure courtesy steve.gb.com
46
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

47
How this works
  • When an unmethylated specific sequence appears in
    the DNA, the enzyme cleaves it
  • When the corresponding methylated sequence
    appears, it doesnt get cleaved and remains
    available for replication
  • The restriction endonucleases only bind to
    palindromic sequences
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