The origin of novel proteins by gene The origin of novel proteins by gene duplication: evolution of translation duplication: evolution of translation

termination factors termination factors

Department of Genetics Department of Genetics St. Petersburg State UniversitySt. Petersburg State University

Galina Galina ZhouravlevaZhouravleva

Part 1.Part 1. MechanismMechanism of translation termination of translation termination

5’ 3’CAPCAP

AUGAUG UAAUAA

Start codon Stop codon

AAAAAAAAAAAAAA

5’ UTR5’ UTR 3’ UTR3’ UTR

RecyclingRecycling

InitiationInitiation ElongationElongation Termination

Main steps in eukaryotic translationMain steps in eukaryotic translation

mRNA

5’ 3’CAPCAP

AUGAUG UAAUAA

Start codon Stop codon

AAAAAAAAAAAAAA

5’ UTR5’ UTR 3’ UTR3’ UTR

InitiationInitiation ElongationElongation Termination

Main steps in eukaryotic translationMain steps in eukaryotic translation

mRNA

Translation factorsTranslation factors::

ProkariotaProkariota::

EukaryotaEukaryota::

IF-1, IF-2, IF-3IF-1, IF-2, IF-3

eIF1, eIF1A, eIF2, eIF1, eIF1A, eIF2, eIF2B, eIF3, eIF4A, eIF2B, eIF3, eIF4A, eIF4B, eIF4E,eIF4B, eIF4E,eIF4G, eIF5eIF4G, eIF5

EF-Tu, EF-Ts, EF-GEF-Tu, EF-Ts, EF-G

eEF1eEF1АА, eEF1, eEF1ВВ, eEF2, eEF2

RF1, RF2, RF3RF1, RF2, RF3

eRF1, eRF3eRF1, eRF3

Е Р А

UGAUGARF1RF1 ( (RF2) + RF3RF2) + RF3

Stop-codon recognition

3’5’

Translation termination in prokaryotes

Translation termination factorsTranslation termination factors - RF- - RF- factors factors ( (RRelease elease FFactors):actors):

RF3RF3 - - GTPase; promotes RF1/2 release GTPase; promotes RF1/2 release (non-essential)(non-essential)

RF2 RF2 (essential) (essential) – – decodes decodes UAA UAA andand UGA UGA

RF1 RF1 (essential) (essential) – – decodes decodes UAA UAA andand UAG UAGClass 1 release factorsClass 1 release factors

Class 2 release factorClass 2 release factor

Е Р А

UGAUGARF2 + RF3RF2 + RF3

36% amino 36% amino acid identityacid identity

Translation termination in eukaryotes

Recycling?Reinitiation?

Е Р А

UGAUGAAAAAAAUUUUUU

eRF1 + eRF3

Peptidyl-tRNA hydrolysis

Е Р А

UGAUGAAAAAAAUUUUUU

GGQGGQ

GTPGTPeRF1eRF3

Е Р А

UGAUGAAAAAAAUUUUUU

GGQGGQ

GTPGTPeRF1eRF3

GTP hydrolysis

PA

B

Е Р А 4G

4E

PAB

UGAUGAAAAAAAUUUUUU 5’

3’

GGQGGQ

GDPGDPeRF3eRF1

Stop-codon recognition

eeRF3RF3 (essential) - GTPase (essential) - GTPase

eeRF1RF1 (essential) (essential) – UAA – UAA,, UAG UAG, UGA, UGAClass 1 release factorClass 1 release factor

Class 2 release factorClass 2 release factor

((RF1 + RF1 + RF2)RF2)

((RFRF3)3)

Part 2.Part 2. Translation termination factors Translation termination factors

RF2 - UAA и UGARF2 - UAA и UGA

RF1 - UAA и UAGRF1 - UAA и UAG

Class 1 release factorsClass 1 release factors

Prokaryota

eeRF1 – UAARF1 – UAA,, UAG UAG, UGA, UGA

Eukaryota Archaea

aaRF1 – RF1 – all 3 stop codons (?)all 3 stop codons (?)

Homologous (30% of identity)

Class Class 22 release factors release factors

No sequence similarity

RFRF33

Prokaryota

eeRFRF33

Eukaryota Archaea

AbsentAbsent

No sequence similarity

Ito et al., 1996

The average similarity plot of RF sequencesThe average similarity plot of RF sequences

A-G – conserved regions

Ito et al., 1996

Comparison of the amino acid sequences of prokaryotic RFs and EF-G of E.coli

Ito et al., 1996

tRNA-protein mimicry hypothesis

Mulitcellular eukaryotes

Phylogenetic tree of aRF1 and eRF1Phylogenetic tree of aRF1 and eRF1

Liu, 2005Inagaki, Doolittle, 2000

Phylogenetic tree of eRF3Phylogenetic tree of eRF3

The phylogenetic tree showing the origin of paralogs The phylogenetic tree showing the origin of paralogs encoding the factors eRF3a and eRF3b in higher eukaryotesencoding the factors eRF3a and eRF3b in higher eukaryotes

DuplicationDuplication

DivergenceDivergence eRF3aeRF3a

eRF3beRF3b

eRF3aeRF3a

eRF3beRF3b

H. sapiensH. sapiens

eRF3eRF3

M. musculusM. musculus

M. musculusM. musculus

H. sapiensH. sapiens

lower eukaryoteslower eukaryotes

Duplication

Difference in the organization of Difference in the organization of GSPTGSPT genes genes

GSPT1 – 15 introns

M.musculus

H.sapiens

GSPT2 – no introns

16 chromosome

16 chromosome

Х chromosome

Х chromosome

5’UTR/1

SplicingSplicing

RetropositionRetroposition

GSPT1 (16 chromosome)

GSPT2 (X chromosome)

PP11 PP22 5’UTR/2

PP22 5’UTR/2

3’UTR

A model of A model of GSPT2GSPT2 origin by reverse transcription of a origin by reverse transcription of a processed processed GSPT1GSPT1 transcript and its reintegration transcript and its reintegration

in X-chromosomein X-chromosome

P1, P2 – promoter sequences

(1-573)X. laevis Sup35

(1-637)Human GSPT1

(1-635)Mouse GSPT1

(1-632)Mouse GSPT2

(1-632)Human GSPT2

(1-685)S. cerevisiaeSup35

14% 13% 57%Amino acid identity between yeast Sup35 and human GSPT1

N M C

+

-

-

NT

+

-

Complementation of S. cerevisiae

SUP35 disruption

eRF3 family

NT

Protein

Yeast proteome

ySup35

mGSPT1

mGSPT2

xSup35

Q+N (%)

10

45

8

4

18

-

100

10

7

14

-

10

100

49

11

G+Y (%)

8

33

10

5

9

N-terminal domain of eRF3 is not conserved in evolutionN-terminal domain of eRF3 is not conserved in evolution

Identity (%)

with yeast

Sup35

with mouse GSPT1

Alpha helix – h, extended strand – e, random coil – c, beta turn - tSOPM (Self-Optimized Prediction Method) - secondary structure prediction method (Geourjon and Deleage, 1994) http://npsa-pbil.ibcp.fr/cgi-bin/

N-terminal domain of eRF3 is not conserved in evolutionN-terminal domain of eRF3 is not conserved in evolution

mGSPT1 -----------------MDPGSGGGGGGGGGGSSSSSDSAPDCWDQTDME------------------ -----------------ccttccccccccccccccccccccccccccccc------------------mGSPT2 -----------------MDLGS-------------SNDSAPDCWDQVDME------------------ -----------------eeecc-------------cccccccccceeeec------------------xSup35 -----------------ITGTTLFPPTWEVLPTLPTPCLTPSAPLIKQLV------------------ -----------------ecccccccccceecccccccccccccchhheee------------------ySup35 MSDSNQGNNQQNYQQYSQNGNQQQGNNRYQGYQAYNAQAQPAGGYYQNYQGYSGYQQGGYQQYNPDAG eccccccccccceeeeccccccccccccccchhhhhhtccccccceecttccttcccttcccccttcc . * :

mGSPT1 APGPGPCGGG---GSGSGSMAAVAEAQR---ENLSAAFSRQLNVNAKPFVPN--- cccccccccc---cccchhhhhhhhhhh---hhhhhhhhhhhcccccccccc---mGSPT2 GPGSAPSGDGIAPAAMAAAEAAEAEAQR---KHLSLAFSSQLNIHAKPFVPS--- cccccccccccchhhhhhhhhhhhhhhh---hhhhhhhhhhccccccccccc---xSup35 YPNPTHPEMDASDSAPDSWEQADMEATE---AQLNNSMA-ALNVNAKPFVPN--- ccccccccccccccccchhhhhhhhhhh---hhhhhhhh-hhhccccccccc---ySup35 YQQQYNPQGGYQQYNPQGGYQQQFNPQGGRGNYKNFNYNNNLQGYQAGFQPQSQG ceeecccttccccccttccceeeccccccccceeeecccccccchettccccctt . . :. . *: * *.

G-stretch

Pab1-interactingregion

QN-stretch Oligopeptide (PQGGYQQ-YN) repeats

Oligopeptide (PQGGYQQ-YN) repeats

Part 3.Part 3. Prionization Prionization of translation termination factor eRF3 in of translation termination factor eRF3 in yeast yeast

Composition of yeast eRF3 (Sup35) Composition of yeast eRF3 (Sup35)

Translation termination

CMN

1 124 254 685

6 33 97

QN

R1 R2 R3 R4 R6PFD

OR

R5

QN: the N-terminal QN-rich stretch. OR: R1-R6 – oligopeptide repeats of the consensus sequence PQGGYQQ-YN (P – proline, Q – glutamine, G – glycine, Y – tyrosine, N – asparagine)

PFD

EF1-A-like domain

Evolutionary comparison of the N-terminal domains of Sup35 Evolutionary comparison of the N-terminal domains of Sup35 proteins from budding and fission yeastproteins from budding and fission yeast

132 D. hansenii (GYQNYNQ)5.5

137 K. lactis

161

(QGYNNAQQ)6

P. methanolica (NRGGYSNYN)5

106 P. pastoris

123

(QGYQXY)4

103

S. cerevisiae (PQGGYQQ-YN)5.5

Z. rouxii (GGYGGY)5

157 Y. lipolytica

121 S. ludwigii

144 C. maltosa

129 C. albicans

(QGGYQGGYQGGY)5

(GYQAYQQYNAQPQQQ)4.5

(GGYQQNYN)6.5

(GGYQQNYNNR)4.5

S. pombe112 No repeats

QN-stretch OR-region

Q(%) N(%)

39 15

43 17

35 30

16 22

37 26

40 12

38 9

45 14

52 15

39 7

No QN-stretch

Yarrowia

Saccharomycodes

Candida

QN OR

Ascomycota

Debaryomyces

Kluyveromyces

Saccharomyces

Zygosaccharomyces

Pichia

Schizosaccharomyces

N-domain

Ancient GTPase

aEF-2

eEF-2

Archaea

Eubacteria

EF-G

EF-GEF-G

RF3

EF-Tu

aEF-1A

eRF3Eukarya

Archaea

eEF-1A eEF-1A

EF-Tu

Eukarya

Evolutionary origin of eRF3Evolutionary origin of eRF3

EF – elongation factor, RF- release factor.

(1-685)Saccharomyces cerevisiae Sup35

N M C

(1-465)Giardia intestinalis Sup35

Part 4.Part 4. Molecular mimicry: Molecular mimicry: translation termination factors as tRNA translation termination factors as tRNA

Ito et al., 1996

tRNA-protein mimicry hypothesis

tRNA-EF-Tu-GTP EF-G-GTP

Molecular Mimicry

tRNA

EF-Tu

(Ramakrishnan 2002)

Macromolecular mimicry in termination Macromolecular mimicry in termination and ribosome recyclingand ribosome recycling

Human eRF1 E. coli RF2 Yeast tRNAPhe

Part 5.Part 5. DuplicationDuplication in the evolutionary history of translation in the evolutionary history of translation elongation and termination factorselongation and termination factors

(Inagaki(Inagaki and Ford, and Ford, 2000) 2000)

A scheme for the evolution of elongation and release factors in Bacteria, Archaea, and Eukarya.

eEF-1A

eEF-2EF-G

EF

EF-Tu

eRF1

RF1

RF2

EF-G

RF3

Hbs1

eRF3EF-Tu

The evolutionary origin of translation The evolutionary origin of translation termination factorstermination factors

EF - elongation factors EF - elongation factors

RF – termination (release) factors RF – termination (release) factors

DuplicationDuplication

DivergenceDivergence

e - eukaryotic

Hbs1