Comparative genomics: functional characterization of new genes and regulatory interactions using computer analysis

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Comparative genomics functional
characterization of new genes and regulatory
interactions using computer analysis

• Mikhail Gelfand
• Institute for Information Transmission
  Problems(The Kharkevich Institute), RAS
• Workshop at the Landau Instiute of Theoretical
  Physics, RAS
• September 27-28, 2007, Moscow

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The genome is decyphered!
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Is it?

• To intercept a message does not mean to
  understand it

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Fragment of a genome (0.1 of E. coli)
A typical bacterial genome several million
nucleotides 600 through 9,000 genes (90 of
the genome encodes proteins)
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Propaganda
sequences in GenBank (genes)
articles in PubMed (experiments)
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More propaganda

• Most genes will never be studied in experiment
• Even in E.coli only 20-30 new genes per year
  (hundreds are still uncharacterized)
• Universally missing genes not a single known
  gene even for 10 reactions of the central
  metabolism. No genes for gt40 reactions overall.
• Conserved hypothetical genes (5-15 of any
  bacterial genome) essential, but unknown
  function.

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The local goal to characterize the genes

• What?
• function (rather, role)
• When?
• regulation (conditions)
• gene expression
• lifetime (mRNA, protein)
• Where?
• Localization
• Cellular/membrane/secreted
• How?
• Mechanism of action
• Specificity, regulation (biochemistry)

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Propaganda-2 complete genomes
2007 gt 1200 bacterial genomes
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The global goal

• to predict the organisms properties given its
  genome
• (plus some additional information, e.g. the
  initial state after cell division)
• and to understand the evolution of
  genomes/organisms

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Haemophilus influenzae, 1995
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Vibrio cholerae, 2000
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The metabolic map, the birds view
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Metabolic pathways, the eagles view
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A submap (metabolism of arginine and proline)
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Approaches

• Similarity gt homology (common origin)
• Homology gt common function
• The Pearson Principle (after Karl
  Pearson)important features are conserved
• functional sites in proteins
• regulatory (protein-binding) sites in DNA
• not necessarily sequences
• structure of protein and RNA
• gene localization on chromosomes
• co-expression of genes
• Allows one to annotate 50-75 of genes in a
  bacterial genome
• Necessary first step, may be automated (to some
  extent)

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but not so simple

• Similarity ? homology
• Low complexity regions, unstructured domains,
  transmembrane segments and other regions with
  non-strandard amino acid composition
• The need for correct similarity measures
• Does homology always follow from the structural
  similarity?
• What is structural similarity?How can it be
  measured?
• Convergent evolution of structures?Independent
  emergence of folds?
• Homology ? same function
• What is the same function?
• Biochemical details and cellular role

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The Fermi principle

• (after Enrico Fermi)
• Purely homology-based annotation boring (nothing
  radically new)
• It turns out, one can predict something
  completely new
• Comparative genomics

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Positional clustering

• Genes that are located in immediate proximity
  tend to be involved in the same metabolic pathway
  or functional subsystem
• caused by operon structure, but not only
• horizontal transfer of loci containing several
  functionally linked operons
• compartmentalisation of products in the cytoplasm
• very weak evidence
• stronger if observed in may unrelated genomes
• May be measured
• e.g. the STRING database/server (P.Bork, EMBL)
• and other sources

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STRING trpB positional clusters
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Functionally dependent genes tend to cluster on
chromosomes in many different organisms
Vertical axis number of gene pairs with
association score exceeding a threshold. Control
same graph, random re-labeling of vertices
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More genomes (stronger links) gt highly
significant clustering
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Fusions

• If two (or more) proteins form a single
  multidomain protein in some organism, they all
  are likely to be tightly functionally related
• Very useful for the analysis of eukaryotes
• Sometimes useful for the analysis of prokaryotes

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STRING trpB fusions
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Phyletic patterns

• Functionally linked genes tend to occur together
• Enzymes with the same function (isozymes) have
  complementary phyletic profiles

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STRING trpB co-occurrence (phyletic patterns)
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Phyletic patterns in the Phe/Tyr pathway
shikimate kinase
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Archaeal shikimate-kinase
Chorismate biosynthesis pathway (E. coli)
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Arithmetics of phyletic patterns
Shikimate dehydrogenase (EC 1.1.1.25) AroE
COG0169 aompkzyqvdrlbcefghsnuj-i--
5-enolpyruvylshikimate 3-phosphate synthase (EC
2.5.1.19) AroA COG0128 aompkzyqvdrlbcefghsnuj-
i--
Chorismate synthase (EC 2.5.1.19)
AroC COG0082 aompkzyqvdrlbcefghsnuj-i--
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Distribution of association scores monotonic
for subunits,bimodal for isozymes
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Comparative analysis of regulation

• Phylogenetic footprinting regulatory sites are
  more conserved than non-coding regions in general
  and are often seen as conserved islands in
  alignments of gene upstream regions
• Consistency filtering regulons (sets of
  co-regulated genes) are conserved gt
• true sites occur upstream of orthologous genes
• false sites are scattered at random

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Riboflavin (vitamin B2) biosynthesis pathway
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5 UTR regions of riboflavin genes from bacteria
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Conserved secondary structure of the RFN-element
Capitals invariant (absolutely conserved)
positions. Lower case letters strongly
conserved positions. Dashes and stars
obligatory and facultative base pairs Degenerate
positions R A or G Y C or U
K G or U B not A V not U.
N any nucleotide. X any
nucleotide or deletion
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RFN the mechanism of regulation

• Transcription attenuation

• Translation attenuation

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Early observation an uncharacterized gene (ypaA)
with an upstream RFN element
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Phylogenetic tree of RFN-elements (regulation of
riboflavin biosynthesis)
no riboflavin biosynthesis
duplications
no riboflavin biosynthesis
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YpaA a.k.a. RibU riboflavin transporterin
Gram-positive bacteria

• 5 predicted transmembrane segments gt a
  transporter
• Upstream RFN element (likely co-regulation with
  riboflavin genes) gt transport of riboflaving or
  a precursor
• S. pyogenes, E. faecalis, Listeria sp. ypaA, no
  riboflavin pathway gt transport of riboflavin
• Prediction YpaA is riboflavin transporter
  (Gelfand et al., 1999)
• Validation
• YpaA transports flavines (riboflavin, FMN, FAD)
  by genetic analysis (Kreneva et al., 2000) by
  direct measurement (Burgess et al., 2006 Vogl et
  al., 2007 )
• ypaA is regulated by riboflavin by microarray
  expression study (Lee et al., 2001)
• via attenuation of transcription (and to some
  extent inhibition of translaition) (Winkler et
  al., 2003)

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Conserved structures of riboswitches (circled
X-ray)
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Mechanisms
gcvT ribozyme, cleaves its mRNA (the Breaker
group)THI-box in plants inhibition of splicing
(the Breaker and Hanamoto groups)
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Characterized riboswitches (more are predicted)
RFN Riboflavin biosynthesis and transport FMN (flavin mononucleotide) Bacillus/Clostridium group, proteobacteria, actinobacteria, other bacteria
THI Biosynthesis and transport of thiamin and related compounds TPP (thiamin pyrophosphate) Bacillus/Clostridium group, proteobacteria, actinobacteria, cyanobacteria, other bacteria, archea (thermoplasmas), plants, fungi
B12 Biosynthesis of cobalamine, transport of cobalt, cobalamin-dependent enzymes Coenzyme B12 (adenosyl-cobalamin) Bacillus/Clostridium group, proteobacteria, actinobacteria, cyanobacteria, spirochaetes, other bacteria
S-boxSAM-IISAM-III Metabolism of methionine and cystein SAM (S-adenosyl- methionine) Bacillus/Clostridium group and some other bacteriaSAM-II (alpha), SAM-III (Streptococci)
LYS Lysine metabolism lysine Bacillus/Clostridium group, enterobacteria, other bacteria
G-box Metabolism of purines purines Bacillus/Clostridium group and some other bacteria
glmS (ribozyme) Synthesis of glucosamine-6-phosphate glucosamine-6-phosphate Bacillus/Clostridium group
gcvT (tandem) Catabolism of glycine glycine Bacillus/Clostridium group
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Properties of riboswitches

• Direct binding of ligands
• High conservation
• Including unpaired regions tertiary
  interactions, ligand binding
• Same structure different mechanisms
  transcription, translation, splicing, (RNA
  cleavage)
• Distribution in all taxonomic groups
• diverse bacteria
• archaea thermoplasmas
• eukaryotes plants and fungi
• Correlation of the mechanism and taxonomy
• attenuation of transcription (anti-anti-terminator
  ) Bacillus/Clostridium group
• attenuation of translation (anti-anti-sequestor
  of translation initiation) proteobacteria
• attenuation of translation (direct sequestor of
  translation initiation) actinobacteria
• Evolution horizontal transfer, duplications,
  lineage-specific loss
• Sometimes very narrow distribution evolution
  from scratch?

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Conserved signal upstream of nrd genes
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Identification of the candidate regulator by the
analysis of phyletic patterns

• COG1327 the only COG with exactly the same
  phylogenetic pattern as the signal
• large scale on the level of major taxa
• small scale within major taxa
• absent in small parasites among alpha- and
  gamma-proteobacteria
• absent in Desulfovibrio spp. among
  delta-proteobacteria
• absent in Nostoc sp. among cyanobacteria
• absent in Oenococcus and Leuconostoc among
  Firmicutes
• present only in Treponema denticola among four
  spirochetes

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COG1327 Predicted transcriptional regulator,
consists of a Zn-ribbon and ATP-cone domains
regulator of the riboflavin pathway (RibX)?
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Additional evidence co-localization

• nrdR is sometimes clustered with nrd genes or
  with replication genes dnaB, dnaI, polA

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Additional evidence co-regulated genes

• In some genomes, candidate NrdR-binding sites are
  found upstream of other replication-related genes
• dNTP salvage
• topoisomerase I, replication initiator dnaA,
  chromosome partitioning, DNA helicase II

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Multiple sites (nrd genes) FNR, DnaA, NrdR
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Mode of regulation

• Repressor (overlaps with promoters)
• Co-operative binding
• most sites occur in tandem (gt 90 cases)
• the distance between the copies (centers of
  palindromes) equals an integer number of DNA
  turns
• mainly (94) 30-33 bp, in 84 31-32 bp 3 turns
• 21 bp (2 turns) in Vibrio spp.
• 41-42 bp (4 turns) in some Firmicutes

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Experimental validations
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Acknowledgements

• Dmitry Rodionov (comparative genomics)
• Andrei Mironov (software)
• Alexei Vitreschak (riboswitches)
• Funding
• Howard Hughes Medical Institute
• Russian Foundation of Basic Research
• RAS, program Molecular and Cellular Biology
• INTAS