Download Nucleic Acids: Revisiting the Central Dogma

Nucleic Acids:
Revisiting the Central Dogma
Kathleen B Hall, Ph.D.
Dept Biochemistry & Molecular
Biophysics
[email protected]
Central Dogma: DNA
DNA
RNA
RNA
protein
protein
Purines
A review of the components of
DNA and RNA.
The nucleobases:
DNA has A, G, T, C.
RNA has A, G, U, C.
Pyrimidines
Each base is attached to a sugar.
DNA (deoxyribonucleic acid) has deoxyribose.
RNA (ribonucleic acid) has ribose.
The base + sugar = nucleoside
Base pairs are hydrogen bonded
3′
5′
Phosphodiester
backbone
links the
nucleotides
together.
In a double-strand
(duplex), strands
are anti-parallel
5′
3′
A-form
B-form
RNA
DNA
DNA
Right-handed
Z-form
DNA
RNA
Left-handed
B-form
A-form
Z-form
In a eukaryotic cell, DNA is not naked.
The double-strand is bound by
histone proteins to create the famous
beads-on-a-string form of chromatin.
The ‘beads’ are nucleosomes, and
they are bundled together to give
chromatin fibers.
Chromatin can be extended (the
transcriptionally active form)
But can also be condensed
(transcriptionally inactive)
such in metaphase chromosomes.
RNA is transcribed from DNA by RNA polymerase.
• Prokaryotes have one RNA polymerase that
transcribes all RNAs
• Eukaryotes have RNA polymerase I, II, and III.
• Pol I transcribes ribosomal RNA (rRNA)
• Pol II transcribes pre-mRNA
• Pol III transcribes tRNA, 5S rRNA (small RNAs)
• Some viruses code for their own RNAP
• Transcribed RNAs are single-stranded, but they fold
to form secondary structure (duplexes) and tertiary
structures.
Pol I and rRNA transcription
23S rRNA
There are three ribosomal RNAs: 28S, 18S, and 5.8S.
Pol II and pre-mRNA transcription
Pre-mRNA splicing
intron
exon
intron
exon
intron
exon
intron
Ribose undergoes unique chemical reactions.
This is an intramolecular transesterification reaction.
Ferré-D'Amaré A R , Scott W G Cold Spring Harb Perspect
Biol 2010;2:a003574
2
1
1
2
Step 1: the 2′ OH of the
branch point Adenosine
attacks the 5′ splice site
of exon1.
2
1
Step 2: the 3′ OH of
exon1 attacks the 3′
splice site.
1
2
1
2
1
The result of the first transesterification reaction is a free 3′ OH on exon1.
The result of the second transesterification reaction is a free intron and
ligated exon1exon2.
These are the reactions that constitute pre-mRNA splicing.
2
5′ splice site
GU
3′ splice site
A
AG
Pre-mRNA splicing
happens in the
SPLICEOSOME
U1, U2, U4, U5, U6
snRNPs*:
Identify the splice sites.
Carry out the splicing
reaction.
Release the ligated
exons.
Recycle.
*small nuclear RiboNucleoprotein Particles
U2 snRNA base pairs with the 3′ splice site
U1 snRNA base pairs with the 5′ splice site
U6 and U2 snRNAs form a new
complex that is thought to be the
catalytic center of the spliceosome
U4 snRNA releases U6 snRNA
~ 94% of human genes are discontinuous, providing opportunity for
alternative splicing and also errors of exon/intron definition.
What mechanisms control alternative splicing?
a) competition for splice sites. A protein could bind at or near a splice site and
block access by the snRNP.
b) developmental or tissue-specific proteins bind to splice sites and block snRNPs
Alternative splicing is the dominant mechanism used to generate protein isoform diversity.
Mutually Exclusive Splicing of the Insect Dscam Pre-mRNA Directed by Competing
Intronic RNA Secondary Structures
Cell 123, 65–73, 2005.
Brenton R. Graveley
Drosophila Dscam encodes 38,016 distinct axon guidance receptors through the
mutually exclusive alternative splicing of 95 variable exons. Importantly, known
mechanisms that ensure the mutually exclusive splicing of pairs of exons cannot
explain this phenomenon in Dscam.
Ribosomes translate the mRNA
A RIBOSOME contains many proteins and three RNAs
A large and
small subunit
Most of the ribosome is RNA
Cross-section of bacterial
70S ribosome.
Blue: small subunit RNA
White: large subunit RNA
Orange: tRNA
Magenta & dark blue:
proteins
Turquoise: nascent
polypeptide chain in exit
tunnel
The catalytic site is made of RNA
Regulation of mRNA expression
1) siRNA
2) miRNA
miRNA (micro RNA) pathway
endogenous
Regulates
gene
expression in
development,
tissues,
cancer, etc
etc
siRNA (small interfering RNA)
pathway
exogenous
Used for knocking
down gene
expression through
the RNAi pathway.
Nuclease
(argonaut protein)
cleaves mRNA
within protein
coding region
(ORF).
Small hairpin
RNAs (shRNA) are
big business for
study of gene
expression.
MicroRNA genes are often clustered in intergenic regions. Others are in introns.
Some pri-miRNAs with their miRNAs in red.
These are human: The
precursor structure is a
box; the black is the
miRNA sequence.
A model of miRNA processing and activity (Ambion website*)
RISC is a complex of proteins with
the antisense strand of miRNA (or
siRNA). The major protein in RISC
is the Argonaut protein. Humans
have 8 isoforms of Argonaut (Ago).
RISC:
RNA Induced Silencing Complex
(*miRNA is also big business)
siRNAs target the ORF. miRNAs (mostly) target an mRNA 3′ untranslated region.
siRNA:mRNA duplexes
are perfect.
miRNA:mRNA duplexes
have mismatches and
internal loops.
The most common
hypothesis is that
translation is repressed but
the mRNA is intact.
There is a recent report
suggesting that the mRNA
is degraded by miRNA
RISC complexes.
The molecular hallmarks of lin-4, the founding member of the microRNA family. Sequence
complementarity between lin-4 (red) and the 3'-untranslated region (UTR) of lin-14 mRNA (blue). lin-4 is
partially complementary to 7 sites in the lin-14 3' UTR; its binding to these sites of complementarity brings
about repression of LIN-14 protein synthesis. [He & Hannon. 2004. Nature Reviews Genetics 5, 522-531]
miRNA expression is analyzed by microarrays. Here’s one commercial example.
DNA
RNA
protein
Nature (1989) 338:217-224
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RNA has template properties. It can store genetic information and
(theoretically) replicate it.
RNA has catalytic properties.
Most biological coenzymes are nucleotides or nucleotide-based
compounds.
Histidine biosynthesis starts with phosphoribosyl pyrophosphate and
ATP
dNTPs are enzymatically synthesized by reduction of rNTPs
dT is formed by 5-methylation of dU
Modern RNAs participate in the ‘most ancient’ cellular properties:
RNA oligonucleotides prime DNA synthesis
tRNA carries genetic message from DNA to ribosome
mRNA, tRNA, and rRNA synthesize proteins
snRNAs are integral spliceosome components
Why RNA?
RNA combines genotype and phenotype.
When it replicates, genetic variation is introduced through
mutation and RNA-catalyzed recombinations to produce new phenotypes.
Darwinian selection occurs by the most efficiently replicating
molecules which will eventually dominate the pool until a new mutant
arises that will take over and so on and so on.
Why not RNA?
RNA is “biochemically inept”.
RNA is not a “plausible pre-biotic molecule” because it won’t have
been present or produced in sufficient quantities to lead to world
domination.
What compounds were present in the prebiotic atmosphere?
H2, H2O, CH4, NH3, CO in a reducing atmosphere
Maybe methane and ammonia came from meteorites?
Both hydrogen cyanide and formaldehyde have been produced in
the lab in spark-discharge experiments (thought to mimic primitive
earth conditions).
HCN and H2CO would have been unstable unless they were
protected, and to be useful for production of other compounds,
they would have to be together in sufficient quantities.
What can you make with HCN and H2CO?
Prebiotic synthesis of purines by self-condensation of HCN (hydrogen cyanide)
DAMN (diaminomaleonitrile) reacts with formamidine or UV light to produce 4aminoimidazole-5-carbonitrile then on to purines.
Formaldehyde
can produce
glycoaldehyde
In a series of
condensation
reactions called
the formose
reaction,
glycoaldehyde
and
formaldehyde
make sugars.
The prebiotic soup would have many ingredients, so how to
decide on the right combination?
The first genetic system was NOT RNA as we know it.
From glycerol
erythritol
from acrolein
aspartate
Other possible
backbones for a
polymer based
only on purines.
pyranosyl
threose
peptide
glycerol
But how to replicate?
What might the first RNA-based replicase have looked like?
RNA was called biochemically incompetent. Is this true?
Did an RNA World ever exist?
Here’s more circumstantial evidence