Сравнительная геномика – окончание

М.Гельфанд «Сравнительная геномика и

системная биология»

БиБи, 4 курс + ШБ, 2 год + ПФУвесна 2013

Сохранение регуляции на больших эволюционных

расстояниях

Genome 2Genome 2Genome 1Genome 1

Set of known sitesSet of known sites PWMPWM

Genome NGenome N

Two major roles of zinc in bacteria

• Structural role in DNA polymerases, primases, ribosomal proteins, etc.

• Catalytic role in metal proteases and other enzymes

Genomes and regulators

nZURFUR family

???

AdcR ?MarR family

pZURFUR family

Regulators and motifs nZUR-nZUR-

AdcRpZUR

TTAACYRGTTAA

GATATGTTATAACATATCGAAATGTTATANTATAACATTTC

GTAATGTAATAACATTAC

TAAATCGTAATNATTACGATTTA

Transporters

• Orthologs of the AdcABC and YciC transport systems

• Paralogs of the components of the AdcABC and YciC transport systems

• Candidate transporters with previously unknown specificity

zinT: regulation

zinT is isolated

fusion: adcA-zinT

E. coli, S. typhi, K. pneumoniae Gamma-proteobacteria

Alpha-proteobacteria

B. subtilis, S. aureus

S. pneumoniae, S. mutans, S. pyogenes, L. lactis, E. faecalis

Bacillus group

Streptococcus group

zinT is regulated by zinc repressors (nZUR-, nZUR-, pZUR)

adcA-zinT is regulated by zinc repressors (pZUR, AdcR) (ex. L.l.)

A. tumefaciens, R. sphaeroides

ZinT: protein sequence analysis

E. coli, S. typhi, K. pneumoniae, A. tumefaciens, R. sphaeroides, B. subtilis

L. lactis

Y. pestis, V. cholerae, B. halodurans

TM Zn AdcA

S. aureus, E. faecalis, S. pneumoniae, S. mutans, S. pyogenes

ZinT

ZinT: summary

• zinT is sometimes fused to the gene of a zinc transporter adcA

• zinT is expressed only in zinc-deplete conditions (regulated by zinc repressors)

• ZinT is attached to cell surface (has a TM-segment)

• ZinT has a zinc-binding domainZinT: conclusions

• ZinT is a new type of zinc-binding component of zinc ABC transporter

Zinc regulation of PHT (pneumococcal histidine triad) proteins of Streptococcus spp.

S. pneumoniae S. equiS. agalactiae

lmb phtD phtE

phtBphtA

lmb phtD

S. pyogenes

phtY

lmb phtD

zinc regulation shown in experiment

Structural features of PHP proteins

• PHT proteins contain multiple HxxHxH motifs

• PHT proteins of S. pneumoniae are paralogs (65-95% id)

• Sec-dependent hydrophobic leader sequences are present at the N-termini of PHT proteins

• Localization of PHT proteins from S. pneumoniae on bacterial cell surface has been confirmed by flow cytometry

PHH proteins: summary

• PHT proteins are induced in zinc-deplete conditions

• PHT proteins are localized at the cell surface

• PHT proteins have zinc-binding motifs

A hypothesis:• PHT proteins represent a new

family of zinc transporters

… incorrect

• Zinc-binding domains in zinc transporters:

EEEHEEHDHGEHEHSH

HSHEEHGHEEDDHDHSHEEHGHEEDDHHHHHDED

DEHGEGHEEEHGHEH

(histidine-aspartate-glutamate-rich)

• Histidine triads in streptococci:

HGDHYHY 7 out of 21

HGDHYHF 2 out of 21

HGNHYHF 2 out of 21

HYDHYHN 2 out of 21

HMTHSHW 2 out of 21

(specific pattern of histidines and aromatic amino acids)

Analyis of PHP proteins (cont’d)

• The phtD gene forms a candidate operon with the lmb gene in all Streptococcus species– Lmb: an adhesin involved in laminin binding, adherence

and internalization of streptococci into epithelial cells

• PhtY of S. pyogenes: – phtY regulated by AdcR– PhtY consists of 3 domains:

PHT internalin H-rich

4 HIS TRIADS LRR IRHDYNHNHTYEDEEGHAHEHRDKDDHDHEHED

PHH proteins: summary-2• PHT proteins are induced in zinc-deplete conditions• PHT proteins are localized at the cell surface• PHT proteins have structural zinc-binding motifs• phtD forms a candidate operon with an adhesin gene • PhtY contains an internalin domain responsible for the

streptococcal invasion

HypothesisPHT proteins are adhesins involved in the attachment of

streptococci to epithelium cells, leading to invasion

Current state• Pht proteins are required for inhibition of complement

deposition on the pneumococcal surface through the recruitment of complement factor H (Oqunniyi et al., 2009)

• Pht proteins may play a role in immune evasion, but the mechanism of function is unlikely to be mediated by factor H binding (Melin et al., 2010)

Zinc and (paralogs of) ribosomal proteins

L36 L33 L31 S14E. coli, S.typhi – – – + –K. pneumoniae – – – – –Y. pestis, V. cholerae – – – + –B subtilis – – + – – + – +S. aureus – – – – – – +Listeria spp. – – – – – +E. faecalis – – – – – – + –S. pne., S. mutans – – – – – –S. pyo., L. lactis – – – – – – +

nZ

UR

pZ

UR

Ad

cR

Zn-ribbon motif (Makarova-Ponomarev-Koonin, 2001)

L36 L33 L31 S14E. coli, S.typhi (–) – (–) + –K. pneumoniae (–) – (–) – –Y. pestis, V. cholerae (–) – (–) + –B subtilis (–) (–) + – (–) + (–) +S. aureus (–) (–) – – – (–) +Listeria spp. (–) (–) – – (–) +E. faecalis (–) (–) – – – (–) + –S. pne., S. mutans (–) (–) – – – (–)S. pyo., L. lactis (–) (–) – – – (–) +

nZ

UR

pZ

UR

Ad

cR

Summary of observations:

• Makarova-Ponomarev-Koonin, 2001:– L36, L33, L31, S14 are the only ribosomal proteins

duplicated in more than one species– L36, L33, L31, S14 are four out of seven ribosomal

proteins that contain the zinc-ribbon motif (four cysteines)

– Out of two (or more) copies of the L36, L33, L31, S14 proteins, one usually contains zinc-ribbon, while the other has eliminated it

• Among genes encoding paralogs of ribosomal proteins, there is (almost) always one gene regulated by a zinc repressor, and the corresponding protein never has a zinc ribbon motif

Bad scenario

Zn-rich conditions

Zn-deplete conditions: all Zn utilized by the ribosomes, no Zn for Zn-dependent enzymes

Regulatory mechanism

ribosomes

Zn-dependentenzymes

R

Sufficient Zn

Zn starvation

R

repressor

Good scenario

Zn-rich conditions

Zn-deplete conditions: some ribosomes without Zn, some Zn left for the enzymes

Prediction … (Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):9912-7.)

… and confirmation (Mol Microbiol. 2004 Apr;52(1):273-83.)

Later: L31 is a depot; S14 and L33 are “failsafe” substitutes (integrity of ribosomes unde zink starvation). Owen et al, 2007: Of seven Zn-ribbon proteins, six are regulated in Streptomycs (also L28, L32, S18)

Метаболический путь синтеза рибофлавина (витамин В2)

ribAribA

ribA ribB

G TP cyclohydrolase II

ribD

ribD

ribG

ribG

P yrim id ine deam inase

3,4-D HB P synthase P yrim id ine reductase

ribHribH R ibo flavin synthase, -cha in

ribEribB

ypaA

R ibo flavin synthase, -chain

GTP

2,5-diam ino-6-hydroxy-4-(5`-phosphoribosylamino)pyrim idine

ribulose-5-phosphate

PENTOSE-PHOSPHATE PATHWAY

PU RINE BIO SYNTHESIS PATHWAY

3,4-dihydroxy-2-butanone-4-phosphate 5-am ino-6-(5`-phosphoribitylam ino)uracil

5-am ino-6-(5`-phosphoribosylamino)uracil

6,7-dimethyl-8-ribityllumazine

Riboflavin

Консервативная последовательность перед генами рибофлавинового пути из очень

разных бактерий 1 2 2’ 3 Add. 3’ Variable 4 4’ 5 5’ 1’ =========> ==> <== ===> -><- <=== -> <- ====> <==== ==> <== <========= BS TTGTATCTTCGGGG-CAGGGTGGAAATCCCGACCGGCGGT 21 AGCCCGTGAC-- 8 4 8 -----TGGATTCAGTTTAA-GCTGAAGCCGACAGTGAA-AGTCTGGAT-GGGAGAAGGATGAT BQ AGCATCCTTCGGGG-TCGGGTGAAATTCCCAACCGGCGGT 19 AGTCCGTGAC-- 8 5 8 -----TGGATCTAGTGAAACTCTAGGGCCGACAGT-AT-AGTCTGGAT-GGGAGAAGGATATG BE TGCATCCTTCGGGG-CAGGGTGAAATTCCCGACCGGCGGT 20 AGCCCGCGA--- 3 4 3 -----AGGATCCGGTGCGATTCCGGAGCCGACAGT-AT-AGTCTGGAT-GGGAGAAGGATGCC HD TTTATCCTTCGGGG-CTGGGTGGAAATCCCGACCGGCGGT 19 AGTCCGTGAC-- 10 4 10 ----–TGGACCTGGTGAAAATCCGGGACCGACAGTGAA-AGTCTGGAT-GGGAGAAGGAAACG Bam TGTATCCTTCGGGG-CTGGGTGAAAATCCCGACCGGCGGT 23 AGCCCGTGAC-- 8 4 8 ----–TGGATTCAGTGAAAAGCTGAAGCCGACAGTGAA-AGTCTGGAT-GGGAGAAGGATGAG CA GATGTTCTTCAGGG-ATGGGTGAAATTCCCAATCGGCGGT 2 AGCCCGCAA--- 3 4 3 ------AGATCCGGTTAAACTCCGGGGCCGACAGTTAA-AGTCTGGAT-GAAAGAAGAAATAG DF CTTAATCTTCGGGG-TAGGGTGAAATTCCCAATCGGCGGT 2 AGCCCGCG---- 7 6 7 --------ATTTGGTTAAATTCCAAAGCCGACAGT-AA-AGTCTGGAT-GGAAGAAGATATTT SA TAATTCTTTCGGGG-CAGGGTGAAATTCCCAACCGGCAGT 6 AGCCTGCGAC-- 11 3 11 ----–CTGATCTAGTGAGATTCTAGAGCCGACAGTTAA-AGTCTGGAT-GGGAGAAAGAATGT LLX ATAAATCTTCAGGG-CAGGGTGTAATTCCCTACCGGCGGT 2 AGCCCGCGA--- 4 4 4 -----ATGATTCGGTGAAACTCCGAGGCCGACAGT-AT-AGTCTGGAT-GAAAGAAGATAATA PN AACTATCTTCAGGG-CAGGGTGAAATTCCCTACCGGTGGT 2 AGCCCACGA--- 3 4 3 -----ATGATTTGGTGAAATTCCAAAGCCGACAGT-AT-AGTCTGGAT-GAAAGAAGATAAAA TM AAACGCTCTCGGGG-CAGGGTGGAATTCCCGACCGGCGGT 3 AGCCCGCGAG-- 5 4 5 ----–TTGACCCGGTGGAATTCCGGGGCCGACGGTGAA-AGTCCGGAT-GGGAGAGAGCGTGA DR GACCTCTTTCGGGG-CGGGGCGAAATTCCCCACCGGCGGT 15 AGCCCGCGAA-- 8 12 9 ----–CCGATGCCGCGCAACTCGGCAGCCGACGGTCAC-AGTCCGGAC-GAAAGAAGGAGGAG TQ CACCTCCTTCGGGG-CGGGGTGGAAGTCCCCACCGGCGGT 3 AGCCCGCGAA-- 5 4 5 -----CCGACCCGGTGGAATTCCGGGGCCGACGGTGAA-AGTCCGGAT-GGGAGAAGGAGGGC AO AATAATCTTCAGGG-CAGGGTGAAATTCCCGATCGGCGGT 2 AGTCCGCGA--- 7 7 7 -----AGGAACCGGTGAGATTCCGGTACCGACAGT-AT-AGTCTGGAT-GGAAGAAGATGAAA DU TTTAATCTTCAGGG-CAGGGTGAAATTCCCGATCGGTGGT 2 AGTCCGCGA--- 13 4 12 -----AGGAACTAGTGAAATTCTAGTACCGACAGT-AT-AGTCTGGAT-GGAAGAAGAGCAGA CAU GAAGACCTTCGGGG-CAAGGTGAAATTCCTGATCGGCGGT 20 AGCCCGCGA--- 3 4 3 -----AGGACCCGGTGTGATTCCGGGGCCGACGGT-AT-AGTCCGGAT-GGGAGAAGGTCGGC FN TAAAGTCTTCAGGG-CAGGGTGAAATTCCCGACCGGTGGT 2 AGTCCACG---- 5 4 5 -------GATTTGGTGAAATTCCAAAACCGACAGT-AG-AGTCTGGAT-GGGAGAAGAATTAG TFU ACGCGTGCTCCGGG-GTCGGTGAAAGTCCGAACCGGCGGT 3 AGTCCGCGAC-- 8 5 8 -----TGGAACCGGTGAAACTCCGGTACCGACGGTGAA-AGTCCGGAT-GGGAGGTAGTACGTG SX -AGCGCACTCCGGG-GTCGGTGAAAGTCCGAACCGGCGGT 3 AGTCCGCGAC-- 8 5 8 -----TTGACCAGGTGAAATTCCTGGACCGACGGTTAA-AGTCCGGAT-GGGAGGCAGTGCGCG BU GTGCGTCTTCAGGG-CGGGGTGAAATTCCCCACCGGCGGT 30 AGCCCGCGAGCG 137 GTCAGCAGATCTGGTGAGAAGCCAGAGCCGACGGTTAG-AGTCCGGAT-GGAAGAAGATGTGC BPS GTGCGTCTTCAGGG-CGGGGCGAAATTCCCCACCGGCGGT 21 AGCCCGCGAGCG 8 4 8 GTCAGCAGATCTGGTCCGATGCCAGAGCCGACGGTCAT-AGTCCGGAT-GAAAGAAGATGTGC REU TTACGTCTTCAGGG-CGGGGTGCAATTCCCCACCGGCGGT 31 AGCCCGCGAGCG 7 5 7 GTCAGCAGATCTGGTGAGAGGCCAGGGCCGACGGTTAA-AGTCCGGAT-GAAAGAAGATGGGC RSO GTACGTCTTCAGGG-CGGGGTGGAATTCCCCACCGGCGGT 21 AGCCCGCGAGCG 11 3 11 GTCAGCAGATCCGGTGAGATGCCGGGGCCGACGGTCAG-AGTCCGGAT-GGAAGAAGATGTGC EC GCTTATTCTCAGGG-CGGGGCGAAATTCCCCACCGGCGGT 17 AGCCCGCGAGCG 8 4 8 GACAGCAGATCCGGTGTAATTCCGGGGCCGACGGTTAG-AGTCCGGAT-GGGAGAGAGTAACG TY GCTTATTCTCAGGG-CGGGGCGAAATTCCCCACCGGCGGT 67 AGCCCGCGAGCG 8 3 8 GTCAGCAGATCCGGTGTAATTCCGGGGCCGACGGTTAA-AGTCCGGAT-GGGAGAGGGTAACG KP GCTTATTCTCAGGG-CGGGGCGAAATTCCCCACCGGCGGT 20 AGCCCGCGAGCG 8 4 8 GTCAGCAGATCCGGTGTAATTCCGGGGCCGACGGTTAA-AGTCCGGAT-GGGAGAGAGTAACG HI TCGCATTCTCAGGG-CAGGGTGAAATTCCCTACCGGTGGT 2 AGCCCACGAGCG 26 9 30 GTCAGCAGATTTGGTGAAATTCCAAAGCCGACAGT-AA-AGTCTGGAT-GAAAGAGAATAAAA VK GCGCATTCTCAGGG-CAGGGTGAAATTCCCTACCGGTGGT 14 AGCCCACGAGCG 11 9 11 GTCAGCAGATTTGGTGAGAATCCAAAGCCGACAGT-AT-AGTCTGGAT-GAAAGAGAATAAGC VC CAATATTCTCAGGG-CGGGGCGAAATTCCCCACCGGTGGT 13 AGCCCACGAGCG 5 4 5 GTCAGCAGATCTGGTGAGAAGCCAGGGCCGACGGTTAC-AGTCCGGAT-GAGAGAGAATGACA YP GCTTATTCTCAGGG-CGGGGTGAAAGTCCCCACCGGCGGT 40 AGCCCGCGAGCG 16 6 16 GTCAGCAGACCCGGTGTAATTCCGGGGCCGACGGTTAT-AGTCCGGAT-GGGAGAGAGTAACG AB GCGCATTCTCAGGG-CAGGGTGAAAGTCCCTACCGGTGGT 25 AGCCCACGAGCG 16 4 27 GTCAGCAGATTTGGTGCGAATCCAAAGCCGACAGTGAC-AGTCTGGAT-GAAAGAGAATAAAA BP GTACGTCTTCAGGG-CGGGGTGCAATTCCCCACCGGCGGT 18 AGCCCGCGAGCG 10 4 10 GTCAGCAGACCTGGTGAGATGCCAGGGCCGACGGTCAT-AGTCCGGAT-GAGAGAAGATGTGC AC ACATCGCTTCAGGG-CGGGGCGTAATTCCCCACCGGCGGT 16 AGCCCGCGAGCA 10 3 11 ---CGCAGATCTGGTGTAAATCCAGAGCCGACGGT-AT-AGTCCGGAT-GAAAGAAGACGACG Spu AACAATTCTCAGGG-CGGGGTGAAACTCCCCACCGGCGGT 34 AGCCCGCGAGCG 6 6 6 GTCAGCAGATCTGGTG 52 TCCAGAGCCGACGGT 31 AGTCCGGAT-GGAAGAGAATGTAA PP GTCGGTCTTCAGGG-CGGGGTGTAAGTCCCCACCGGCGGT 13 AGCCCGCGAGCG 7 3 7 GTCAGCAGATCTGGTGCAACTCCAGAGCCGACGGTCAT-AGTCCGGAT-GAAAGAAGGCGTCA AU GGTTGTTCTCAGGG-CGGGGTGCAATTCCCCACCGGCGGT 17 AGCCCGCGAGCG 7 9 7 GTCAGCAGATCCGGTGAGAGGCCGGAGCCGACGGT-AT-AGTCCGGAT-GGAAGAGGACAAGG PU AAACGTTCTCAGGG-CGGGGTGCAATTCCCCACCGGCGGT 19 AGCCCGCGAGCG 19 4 18 GTCAGCAGACCCGGTGTGATTCCGGGGCCGACGGTCAC-AGTCCGGATGAAGAGAGAACGGGA PY TAACGTTCTCAGGG-CGGGGTGCAACTCCCCACCGGCGGT 19 AGCCCGCGAGCG 15 4 16 GTCAGCAGACCCGGTGTGATTCCGGGGCCGACGGTCAT-AGTCCGGATGAAGAGAGAGCGGGA PA TAACGTTCTCAGGG-CGGGGTGAAAGTCCCCACCGGCGGT 19 AGCCCGCGAGCG 14 4 13 GTCAGCAGACCCGGTGCGATTCCGGGGCCGACGGTCAT-AGTCCGGATAAAGAGAGAACGGGA MLO TAAAGTTCTCAGGG-CGGGGTGAAAGTCCCCACCGGCGGT 16 AGCCCGCGAGCG 8 5 8 GTCAGCAGATCCGGTGTGATTCCGGAGCCGACGGTTAG-AGTCCGGAT-GAAAGAGGACGAAA SM AAGCGTTCTCAGGG-CGGGGTGAAATTCCCCACCGGCGGT 34 AGCCCGCGAGCG 8 3 8 GTCAGCAGATCCGGTCGAATTCCGGAGCCGACGGTTAT-AGTCCGGAT-GGAAGAGAGCAAGC BME GCTTGTTCTCGGGG-CGGGGTGAAACTCCCCACCGGCGGT 17 AGCCCGCGAGCG 10 15 10 GTCAGCAGATCCGGTGAGATGCCGGAGCCGACGGTTAA-AGTCCGGAT-GGAAGAGAGCGAAT BS ATCAATCTTCGGGG-CAGGGTGAAATTCCCTACCGGCGGT 18 AGCCCGCGA--- 5 4 5 -----AGGATTCGGTGAGATTCCGGAGCCGACAGT-AC-AGTCTGGAT-GGGAGAAGATGGAG BQ GTCTATCTTCGGGG-CAGGGTGAAAATCCCGACCGGCGGT 27 AGCCCGCGA—-- 3 5 3 -----AGGATTTGGTGTGATTCCAAAGCCGACAGT-AT-AGTCTGGAT-GGGAGAAGATGGAG BE ATTCATCTTCGGGG-CAGGGTGAAATTCCCGACCGGCGGT 20 AGCCCGCGA--- 3 4 3 -----AGGATCCGGTGCGAGTCCGGAGCCGACAGT-AT-AGTCTGGAT-GGGAGAAGATGAAG CA AATGATCTTCAGGG-CAGGGTGAAATTCCCTACCGGCGGT 2 AGCCCGCGAG-- 3 4 3 ----TATGATCCGGTTTGATTCCGGAGCCGACAGT-AA-AGTCTGGAT-GAAAGAAGATATAT DF GAAGATCTTCGGGG-CAGGGTGAAATTCCCTACCGGCGGT 2 AGCCCGCG---- 6 4 6 -------GATTTGGTGAGATTCCAAAGCCGACAGT-AA-AGTCTGGAT-GAGAGAAGATATTT EF GTTCGTCTTCAGGGGCAGGGTGTAATTCCCGACCGGTGGT 3 AGTCCACGAC-- 5 3 5 ----ATTGAATTGGTGTAATTCCAATACCGACAGT-AT-AGTCTGGAT—-AAAGAAGATAGGG LLX AAATATCTTCAGGG-CACCGTGTAATTCGGGACCGGCGGT 21 ACTCCGCGAT-- 4 4 4 ----–TTGAAGCAGTGAGAATCTGCTAGCGACAGT-AA-AGTCTGGAT-GGAAGAAGATGAAC LO GTTCATCTTCGGGG-CAGGGTGCAATTCCCGACCGGTGGT 3 AGTCCACGAT-- 3 10 3 ----TTGACTCTGGTGTAATTCCAGGACCGACAGT-AT-AGTCTGGAT-GGGAGAAGATGTTG PN AAGAGTCTTCAGGG-CAGGGTGAAATTCCCGACCGGCGGT 125 AGTCCGTG---- 3 4 3 -------GATGTGGTGAGATTCCACAACCGACAGT-AT-AGTCTGGAT-GGGAGAAGACGAAA ST AAGTGTCTTCAGGG-CAGGGTGTGATTCCCGACCGGCGGT 14 AGTCCGCG---- 3 4 3 -------GATGTGGTGTAACTCCACAACCGACAGT-AT-AGTCTGGAT-GAGAGAAGACCGGG MN AAGTGTCTTCAGGG-CAGGGTGAGATTCCCGACCGGCGGT 104 AGTCCGCG---- 3 4 3 -------GATGTGGTGAAATTCCACAACCGACAGT-AA-AGTCTGGAT-GGGAGAAGACTGAG SA ATTCATCTTCGGGG-TCGGGTGTAATTCCCAACCGGCAGT 6 AGCCTGCGAC-- 11 3 11 ----–CTGATCTAGTGAGATTCTAGAGCCGACAGT-AT-AGTCTGGAT-GGGAGAAGATGGAG AMI TCACAGTTTCAGGG-CGGGGTGCAATTCCCCACTGGCGGT 14 AGCCCGCGC--- 5 5 5 ------TGATCTGGTGCAAATCCAGAGCCAACGGT-AT-AGTCCGGAT-GGAAGAAACGGAGC DHA ACGAACCTTCGAGG-TAGGGTGAAATTCCCGACCGGCGGT 20 AGCCCGCAAC-- 11 4 11 --CGACTGACTTGGTGAGACTCCAAGGCCGACGGT-AT-AGTCCGGAT-GGGAGAAGGTACAA FN AATAATCTTCGGGG-CAGGGTGAAATTCCCGACCGGTGGT 2 AGTCCACG---- 4 6 4 -------GATTTGGTGAAATTCCAAAACCGACAGT-AG-AGTCTGGAT-GAGAGAAGAAAAGA GLU ---TGTTCTCAGGG-CGGGGCGAAATTCCCCACCGGCGGT 28 AGCCCGCGAGCG 10 4 10 GTCAGCAGATCCGGTTAAATTCCGGAGCCGACGGTCAT-AGTCCGGAT-GCAAGAGAACC---

Консервативная вторичная структура RFN-элемента

NNNNyYYUC

NNNNrRRAG

NgGGNcCC

rgGGxc

ARRgxuAG

GRCCYG

AcCG

AGCCRGY

GG YRCC

GRYBy CYRVrG N

YGNaA N U U x N

Nx

AGU

UrN A g

Y

variab lestem -loop

additionalstem -loop

3 4

2

1

5

5 ’ 3 ’

u K NRA

xK

*

****

Capitals: invariant (absolutely conserved) positions.

Lower case letters: strongly conserved positions.

Dashes and stars: obligatory and facultative base pairs

N: any nucleotide. X: any nucleotide or deletion

RFN: механизм регуляции• Transcription attenuation

• Translation attenuation

… и еще перед одним геном (ypaA)

NNNNyYYUC

NNNNrRRAG

NgGGNcCC

rgGGxc

ARRgxuAG

GRCCYG

AcCG

AGCCRGY

GG YRCC

GRYBy CYRVrG N

YGNaA N U U x N

Nx

AGU

UrN A g

Y

variab lestem -loop

additionalstem -loop

3 4

2

1

5

5 ’ 3 ’

u K NRA

xK

*

****

цветные стрелки – гены пути

желтые стрелки – ypaA, ген с неизвестной функцией

черные стрелки – регуляторный элемент

YpaA/RibU: транспортёр рибофлавина

• 5 предсказанных ТМ-сегментов => потенциальный транспортёр

• регуляторный RFN-элемент => ко-регуляция с генами метаболизма рибофлавина => транспорт рибофлавина или предшественника

• S. pyogenes, E. faecalis, Listeria: есть ypaA, нет генов биосинтеза рибофлавина => транспорт рибофлавина

Предсказание: YpaA – рибофлавиновый транспортёр (Gelfand et al., 1999)

Проверка:• генетический анализ

(Кренева и др., 2000)

• биохимический эксперимент (Burgess et al., 2006)

Биотиновый

транспортер BioY

Метаболическая реконструкция пути биосинтеза тиамина (витамин В1)

= thiN (confirmed)

(Gram-positive bacteria)

(Gram-negative bacteria)

Transport of HMPTransport of HET

yuaJ(=thiT) тиаминовый транспортер (возможно,

H+-зависимый) в фирмикутах

• 6 предсказанных трансмембранных сегментов• Почти всегда регулируется THI-рибопереключателями• Встречается в геномах, в которых отсутствует

тиаминовый путь (Streptococcus spp.);• В B. cereus импорт тиамина сопряжен с током протонов

(Arch. Microbiol., 1977)

• Почти всегда регулируются THI-рибопереключателями• Не встречаются в геномах, в которых отсутствует

тиаминовый путь• Всегда встречаются вместе с thiD и thiE• В ряде геномов (Pasteurellacee, Brucella некоторые

фирмикуты) встречаются в отсутствие thiC

thiX-thiY-thiZ и ykoF-ykoE-ykoD-ykoC: предсказанные АТФ-зависимые транспортеры HMP

Co и Ni

• ко-локализация (хромосомные локусы)– транспортеры Ni –

с генами никель-зависимых ферментов

– транспортеры Co – с генами синтеза кобаламина

• ко-регуляция– транспортеры Ni –

фактор транскрипции NikR

– транспортеры Co – рибопереключатель В12

A

A

A

AA

AA

CGd

a

aa

a

a

ktk

h

CC

c

C

C

GG

G

GGG

G

GT

M

Y

K

y

c

c G

g

g G

G

G YG

tg

g

g

gN

RN

N

NN

r

r

r

g

g C

c

c T

C

C G

CC

a

ta N

B 12 box

P 0

5' 3'

P 1

P 4 V S

B I IB I

P 5 P 6

P 2

N

A dd- I

F acultative stem- loop

A dd- I I

The group

Bacillus/Clostridium

Other taxonomic groups

-proteobacteria

base stem

CGh

G

d

yc c

C C

P 3

Дмитрий Родионов Thomas Eitinger

Пять семейств транспортеров

Новое семейство транспортеров Co и Ni

Структура локусов

B12-элемент сайт связывания NikRгены

Проверка: тест на транспорт ионов

Co Co

Co

Ni

Ni

Ni

Структура: слишком много компонентов

Биотиновый

транспортер BioY

• АТФаза BioM ~ CbiO = NikO

• Пермеаза BioN ~ CbioQ = NikQ

Для транспорта достаточно компонент МN (первый пример такого

АВС-транспортера)

cbiMNQO

cbiMNQ

cbiMN

cbiM

контроль

BioY тоже достаточно(даже в геномах, содержащих

BioMN); у BioMNY более крутая кинетика

Верхушка айсберга?

http://images.google.ru/imgres?imgurl=http://bsrweb.burnham.org/public/andrei.jpg&imgrefurl=http://bsrweb.burnham.org/metadot/index.pl%3Fid%3D2568%26isa%3DCategory%26op%3Dshow&usg=__QG9V5SCdwHrXMjnI8KsXEMw0WvY=&h=672&w=537&sz=115&hl=ru&start=2&um=1&itbs=1&tbnid=wDNzAzP3RqnHbM:&tbnh=138&tbnw=110&prev=/images%3Fq%3Dandrei%2Bosterman%26um%3D1%26hl%3Dru%26newwindow%3D1%26sa%3DN%26rlz%3D1T4GFRC_ruRU212RU212%26tbs%3Disch:1

Экспериментальные подтверждения

RibU: рибофлавин ThiT: тиамин FolT: фолат (ср. BioY)

Универ-сальный «энергети-ческий комплекс» + компоненты, определя-ющие специфич-ность

P Zhang et al. Nature 000, 1-4 (2010) doi:10.1038/nature09488

The overall structure of RibU.

NAD Metabolism: from E. coli to…

Nicotinate

Nicotinamide

Nicotinamide

NA Ribose

Transporter (pnuC)

NicotinamideAmidohydrolase

(pncA)

Nicotinamide Phospho-ribosyl transferase

(pncB)

Aspartate Oxidase(nadB)

QuinolinateSynthase (nadA)

Nicotinamide RiboseKinase (nadR)

Imino-Asp

Nm Ribose

Asp

NaMN AdenylylTransferase (nadD)

NAD Synthase (nadE)

NAD Kinase

NMN AdenylylTransferase (nadR)

Quinolinate Phospho-ribosyl Transferase

(nadC)

NADNaAD

NADP

Quinolinate

NMNNaMN

Universal and essential enzymeMissing genePredicted by chromosomal clusteringExperimentally verified

NAD Metabolism: …Man

Nicotinamide phospho-ribosyl transferase

5 StepAerobicPathway

Trp

Niacin

Nicotinate

Nicotinamide

Nicotinamide

Nm Ribose

Transporter (?)

NicotinamideAmidohydrolase

(pncA)

Nicotinamide Phospho-ribosyl transferase

(pncB)

Aspartate Oxidase(nadB)

QuinolinateSynthase (nadA)

Nicotinamide RiboseKinase (?)

Imino-Asp

Nm Ribose

Asp

NaMN AdenylylTransferase (hsPNAT)

NAD Synthase

NAD Kinase

NMN AdenylylTransferase (hsPNAT)

Quinolinate Phospho-ribosyl Transferase

NADNaAD

NADP

Quinolinate

NMNNaMN

Found in three newly

sequenced bacteria

Found in H.ducrei

Dual specificity (NaMN and NMN) Missing gene Projected from bacterial NadD Activates a cancer drug