resolving the dynamics of gene action at atomic through … · 2020. 10. 9. · resolving the...
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Resolving the dynamics of gene action at atomic through organismal scales
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http://dordognesnicest.com/the-cave-of-rouffignac/ ≈12000 BCE
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Sequencing mastodon and woolly mammoth genomes
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Central Dogma of Molecular Biology
• DNA makes RNA makes protein – DNA -> DNA is replication
• DNA bases (4 standard): G, A, T, C
– DNA -> RNA is transcription • RNA bases (4 standard) : G, A, U, C
– RNA -> protein is translation • Protein amino acids (20 standard):
A,C,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y
• Modified dogma – RNA -> DNA is reverse transcription
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Oscar Miller (1925-2012)
Miller, O.L., Jr., Hamkalo, B.A., and Thomas, C.A., Jr.
(1970). Science 169, 392-395.
RNA Pol
Visualization of bacterial genes in action
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Double-strand DNA
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Single-strand DNA
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Translation of DNA – 3 forward
reading frames
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Translation of DNA –
first reading frame
IFIHAVESEENFXRTHERITISXYSTANDINGXNTHESHXXLDERSXFGIANTS
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Sir Isaac Newton to Robert Hooke 5 Feb 1675
http://digitallibrary.hsp.org/index.php/Detail/Object/Show/object_id/9285
IF I HAVE SEEN FURTHER IT IS BY
STANDING ON THE SHOULDERS OF GIANTS
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http://www.nature.com/nature/journal/v456/n7218/extref/nature07517-s1.pdf
Figure S1. a. Structure of the
reversible terminator 3’-O-
azidomethyl 2’-deoxythymine
triphosphate (T) labelled with
a removable fluorophore. b.
Structure of the incorporated
nucleotide after removal of
the fluorophore and
terminator group. Each of the
four nucleotides have an
equivalent structure to the
one shown here, except for
the different base and a
corresponding base-specific
fluor.
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http://array.mc.vanderbilt.edu/Sequencing_slides-Intro_Seminar.pdf
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http://www.nature.com/nature/journal/v456/n7218/extref/nature07517-s1.pdf
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http://www.nature.com/nature/journal/v456/n7218/extref/nature07517-s1.pdf
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http://www.nature.com/nature/journal/v456/n7218/extref/nature07517-s1.pdf
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http://array.mc.vanderbilt.edu/Sequencing_slides-Intro_Seminar.pdf
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Fig.1 Cycles 9 through 13 of stack 87 for the variant MFN_R364W. Colors are
red: C, green: G, blue: T. The expected sequence is TCTGG (blue, red, blue,
green, green). The location of the variant is circled.
http://www.openwetware.org/wiki/User:Andrea_Loehr
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Fig.2 Cycles 11 through 15 of stack 27 for the control sequence for
variant MFN_R364W. Colors are red: C, green: G, blue: T. The expected
sequence is TCCGG (blue, red, red, green, green). The location of the
control is circled.
http://www.openwetware.org/wiki/User:Andrea_Loehr
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Next Steps
Next, we will automate the image processing with a shell script and
ImageMagick. We will install and run the Swift software on BOINC.
http://www.openwetware.org/wiki/User:Andrea_Loehr
http://www.imagemagick.org/script/index.phphttp://sgenomics.org/swift/http://boinc.berkeley.edu/
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Contact Info
Andrea Loehr
Harvard-Smithsonian Center for Astrophysics
Cambridge, MA, USA
Email me through OpenWetWare
I work at the Harvard Smithsonian Center for
Astrophysics. I learned about OpenWetWare through the
Church Lab, and I've joined because I have started to
work on a project with the Personal Genome Project
(PGP). Our goal is to set up a volunteer computing project
PersonalGenomes@Home, analogous to seti@home.
http://www.openwetware.org/wiki/User:Andrea_Loehr
http://www.openwetware.org/wiki/Special:EmailUser/Andrea_Loehrhttp://www.openwetware.org/wiki/Special:EmailUser/Andrea_Loehrhttp://www.cfa.harvard.edu/http://www.cfa.harvard.edu/http://www.openwetware.org/wiki/OpenWetWarehttp://www.openwetware.org/wiki/Church_Labhttp://www.personalgenomes.org/http://www.personalgenomes.org/http://www.openwetware.org/wiki/PersonalGenomes@Home
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Levels of genetic control
• Chromatin structure
• Transcriptional control
• Post-transcriptional control
– RNA capping, intron processing, editing, polyadenylation
– RNA export
– RNA stability
• Translational control
• Post-translational control
– Protein modification, splicing, stability
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Translational control
• Global control
– Changes in the translational machinery or masking of mRNA
• mRNA specific control
– Cis-acting sequences affecting translation
• Binding to factors
• Binding to miRNA
• Effects on ribosome behavior at the level of initiation, elongation or termination of translation
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Galagan et al. 2003 Nature 422, 859-868
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Growth of y14, wild-type and upf1 strains –
DELETED THE MOVIE ON THIS SLIDE
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Common and rare mRNA structures
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Prevalence of eukaryotic upstream open reading frames (uORFs)
• Estimates are that 15-53% of mRNAs contain uORFs • Large numbers of fungal mRNAs contain upstream
AUGs (Galagan et al., Nature 2005) – Aspergillus nidulans
• 21% EST 5’UTRs – Neurospora crassa
• 22% EST 5’UTRs
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Nascent peptide control of ribosome movement
• Arg-specific ribosome stalling mediated by the nascent AAP (arginine attenuator peptide)
• AAP-mediated ribosome stalling governs mRNA stability through the nonsense-mediated mRNA decay (NMD) pathway
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Origins of studies on arginine metabolism in Neurospora
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AAP-mediated stalling at the uORF termination codon blocks access to the downstream initiation codon
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The full-length AAP (AAP24) stalls ribosomes
with uORF the stop codon in the A-site of the
ribosome peptidyl transferase center (PTC)
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Evolutionary
conservation of
the AAP
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Evolutionary conservation of the AAP
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Oscar Miller (1925-2012)
Miller, O.L., Jr., Hamkalo, B.A., and Thomas, C.A., Jr.
(1970). Science 169, 392-395.
RNA Pol
Visualization of bacterial genes in action
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Eukaryotic mRNA translation
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Eukaryotic mRNA translation
Afonina, Z.A., et al. (2014). Nucleic Acids Res 42, 9461-9469
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Neurospora ribosome structure – Sachs and Beckmann labs
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Eukaryotic translation initiation
-6 +4 +1 -1
-6 +4 +1 -1
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Eukaryotic initiation from near-cognate codons
AAAn GCCACCAUGG
CUGG
UUGG
GUGG
AAGG
ACGG
AGGG
AUAG
AUCG
AAAG
40S
eIF4F
eIF2
Met
Initiation codon Mammalian efficiency (%)
AUG 100.0
CUG 19.5
GUG 9.2
ACG 6.6
AUA 3.3
AUU 3.2
UUG 1.9
AUC 1.7
AAG 0.2
AGG 0.1 Ivanov, I.P., Loughran, G., Sachs, M.S., and Atkins, J.F.
(2010). Proc Natl Acad Sci U S A 107, 18056.
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Determining the hierarchy of near-cognate codons in N. crassa in vivo
Wei, J., Wu, C., and Sachs, M. S. (2013) J. Biol. Chem. 288, 9549-9562
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CUG and GUG are the best near-cognate codons in vivo
Wei, J., Wu, C., and Sachs, M. S. (2013) J. Biol. Chem. 288, 9549.
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Wei, J., Wu, C., and Sachs, M. S. (2013) J. Biol. Chem. 288, 9549-9562
Prevalence and relevance of
potential initiation at near-cognate
codons in N. crassa
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CPC1
Pezizomycotina cpc-1 homologs all share a
conserved N-terminal extension with a near cognate
start codon in an optimal context
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N. crassa cpc-1 (NCU04050) –
the homolog of S. cerevisiae GCN4
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Conservation of cpc-1 5’UTR coding capacity in Basidiomycota
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Ribosome
profiling
Ribosome profiling: Ingolia and Weissman
Ingolia NT, Brar GA, Rouskin S, McGeachy AM, & Weissman JS (2012) The ribosome
profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-
protected mRNA fragments. Nat Protoc 7(8):1534-1550
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Ribosome
profiling
Ribosome profiling: eIF1
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1st nt in frame 1 1st nt in frame 2 1st nt in frame 3
Ribosome profiling: cpc-1
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Genetic variation
IFIHAVESEENFXRTHERITISXYSTANDINGXNTHESHXXLDERSXFGIANTS
IFIHAVESEENFXRTHERITISXYSTANDINGXNTHESHXXLDERSXF--ANTS
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Fungi that control animal behavior
http://smithsonianscience.si.edu/wordpress/wp-content/uploads/2012/08/modern-day-ant2.jpg
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Ophlocordyceps takes over the ant to create a zombie ant
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Analyses of gene expression patterns in fungus and ant zombie to understand gene action
de Bekker, C., Ohm, R.A., Loreto, R.G., Sebastian, A., Albert, I., Merrow, M.,
Brachmann, A., and Hughes, D.P. (2015). Gene expression during zombie ant
biting behavior reflects the complexity underlying fungal parasitic behavioral
manipulation. BMC Genomics 16, 620.
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Zhai, B., Wu, C., Wang, L., Sachs, M.S., and Lin, X. (2012). The antidepressant
sertraline provides a promising therapeutic option for neurotropic cryptococcal
infections. Antimicrob Agents Chemother 56, 3758-3766.