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TRANSCRIPTIONAL AND TRANSLATIONAL CONTROL MECHANISMS IN NUTRIENT REGULATION OF GENE EXPRESSION
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Alan
G. Hinnebusch, PhD, Head,
Section on Nutrient Control of Gene Expression Hongfang
Qiu, PhD, Staff Scientist Vera Cherkasova,
PhD, Senior Research Fellow Jinsheng
Dong, PhD, Senior Research Assistant Fan Laxminarayana
Burela, PhD, Postdoctoral Fellow Chhabi
Govind, PhD, Postdoctoral Fellow Christie
Hamilton, PhD, Postdoctoral Fellow Antonina
Jivotovskaya, PhD, Postdoctoral Fellow Soon-ja
Kim, PhD, Postdoctoral Fellow Klaus
Nielsen, PhD, Postdoctoral Fellow Leos
Valasek, PhD, Postdoctoral Fellow Sungpil
Yoon, PhD, Postdoctoral Fellow Emily
Ashcraft, BS, Predoctoral Fellow Stephen
Blakely, BS, Predoctoral Fellow Yuen Nei Cheung, BS, Predoctoral Fellow Anna
Krueger, BS, Predoctoral Fellow Evelyn
Sattlegger, PhD, Adjunct Scientist Cuihua Hu, Special Volunteer |
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We
study regulatory mechanisms in the yeast Saccharomyces cerevisiae that
stimulate transcription of amino acid, vitamin, and purine biosynthetic genes
in response to nutrient limitation. Translation of the transcriptional
activator GCN4 is stimulated in starved cells by a mechanism involving short
open reading frames (uORFs) in the mRNA leader and phosphorylation of
initiation factor eIF2. Bound to GTP, the eIF2 delivers initiator tRNAiMet
to the 40S ribosome. Phosphorylation of eIF2 by GCN2 inhibits formation of
the eIF2-GTP-tRNAiMet ternary complex (TC), reducing
general protein synthesis but stimulating translation of GCN4. We are
analyzing the interactions of eIF2 with other initiation factors and the 40S
ribosome, interactions that promote TC recruitment, ribosomal scanning, and recognition
of AUG codons during general and GCN4-specific translation. We also study the
regulation of GCN2 kinase activity by uncharged tRNA (the starvation signal),
a stimulatory protein (GCN1) that associates with GCN2 on translating
ribosomes, and an inhibitory protein (YIH1) that competes with GCN2 for
complex formation with GCN1. We also analyze the co-activators required for
transcriptional activation by GCN4 to define the molecular program for the
recruitment of chromatin remodeling enzymes and adaptor proteins that deliver
TATA-binding protein, other general transcription factors, and RNA polymerase
to target genes. Analysis
of the multifactor complex in pre-initiation complex assembly, scanning, and
AUG selection Nielsen, Valasek,
Jivotovskaya, Dong, Hinnebusch Assembly
of the 80S translation initiation complex is a multistep process involving a
large number of soluble eukaryotic initiation factors (eIFs). According to
current models, the TC binds to the 40S ribosome with the help of eIFs 1, 1A,
and 3. The 43S pre-initiation complex thus formed interacts with mRNA in a
manner stimulated by eIF4F and poly(A)-binding protein. The resulting 48S
complex scans the mRNA until the Met-tRNAiMet base-pairs
with the AUG start codon. On AUG recognition, the eIF5 stimulates GTP
hydrolysis by eIF2, the eIFs are ejected, and the 60S subunit joins with the
40S-Met-tRNAiMet-mRNA complex in a reaction stimulated
by eIF5B. We are probing the relative importance of eIFs -1, -1A, and -3 in
the recruitment of TC and mRNA to the 40S ribosome, scanning, and AUG
selection in vivo by generating mutations in these factors and
examining the consequences on the rate of translation initiation, 43S/48S
complex assembly, and GCN4 translational control in living cells. We
showed previously that eIF3 contains five subunits and exists in a
multifactor complex (MFC) with eIFs 1, 5, and the eIF2-GTP-Met-tRNAiMet
TC. We demonstrated that MFC integrity depends on simultaneous interaction of
the eIF5 C-terminal domain (CTD) with eIF1, eIF3c/NIP1, and eIF2-beta and
that disrupting these interactions by the tif5-7A mutation in the
eIF5-CTD impairs translation initiation and cell growth. We found that the
TIF32-CTD also interacts directly with eIF2-beta and that overexpressing a
form of TIF32 (TIF32-del6) lacking CTD exacerbates the translation initiation
defect of the tif5-7A mutation. Recently, using a new technique we had
developed for cross-linking the components of 43S/48S pre-initiation
complexes in living yeast cells, we demonstrated that overexpressing
TIF32-del6 in a tif5-7A mutant reduces the level of eIF2 binding to
40S subunits in vivo. Our data indicate that the two independent
eIF2-eIF3 contacts in the MFC mediated by eIF5-CTD and TIF32-CTD have
additive stimulatory effects on the efficiency of TC recruitment and the rate
of translation initiation in vivo (Nielsen et al., 2004). Reducing
the rate of TC binding to 40S ribosomes is expected to constitutively
derepress GCN4 translation (Gcd- phenotype).
We previously observed this phenotype for a mutant lacking the CTD of eIF1A
and demonstrated that truncated eIF1A is defective for TC binding to 40S
subunits in a purified system (Olsen et al., 2003). By contrast, we
did not observe a Gcd- phenotype in tif5-7A
cells overexpressing the CTD-less form of TIF32 from a high-copy plasmid
(hc TIF32-del6) despite the reduced levels of 40S-associated TC. We
thus inferred that eIF2-eIF3 contacts in the MFC also contribute to functions
downstream of TC recruitment, such as scanning and AUG recognition, and that
defects in these processes suppress the effects of reduced TC recruitment on GCN4
translation. In support of this conclusion, we found that the tif5-7A mutation
produces a Gcn- phenotype, indicating a reduced level of GCN4
translation in starved cells when eIF2-alpha is phosphorylated by GCN2.
The Gcn- phenotype is suppressed by hc TIF32-del6,
indicating that tif5-7A and hc TIF32-del6 have opposing effects
on GCN4 translation. Interestingly, we found that the prt1-1 mutation
in the b-subunit of eIF3 also produces a Gcn- phenotype and leads to accumulation of 48S
complexes containing TC, mRNA, and all relevant eIFs under nonpermissive
conditions where translation initiation is impaired. Thus, eIF3 is critically
required in vivo for one or more post-assembly functions of the 48S
complex. Analysis of the expression of GCN4-lacZ reporters with
different configurations of uORFs suggests that the Gcn- phenotype in the prt1-1 mutant arises
from a delay in scanning by reinitiating 40S ribosomes between uORFs 1 and 4,
compensating for the reduced rate of TC binding produced by eIF2-alpha
phosphorylation. We are testing this hypothesis by examining mutations in the
eIF4 group of factors for a Gcn- phenotype, as these proteins were previously
implicated in ribosomal scanning in vitro. Interestingly, we found
that the prt1-1 mutation leads to more stringent selection of AUG
triplets as start codons during the scanning processes but does not seem to
impair the rate of 40S-60S subunit joining at the last step of the initiation
pathway. We therefore conclude that eIF3 has critical functions in both
scanning and AUG recognition following assembly of 48S complexes (Nielsen et
al., 2004). Nielsen KH, Szamecz B, Valasek L,
Jivotovskaya A, Shin B, Hinnebusch AG. Functions of eIF3 downstream of 48S
assembly impact AUG recognition and GCN4 translational control. EMBO
J 2004;23:1166-1177. Olsen DS, Savner EM, Mathew A, Zhang F,
Krishnamoorthy T, Phan L, Hinnebusch AG. Domains of eIF1A that mediate
binding to eIF2, eIF3 and eIF5B and promote ternary complex recruitment in
vivo. EMBO J 2003;22:193-204. Valasek L, Mathew AA, Shin BS, Nielsen KH,
Szamecz B, Hinnebusch AG. The yeast eIF3 subunits TIF32/a, NIP1/c, and eIF5
make critical connections with the 40S ribosome in vivo. Genes Dev
2003;17:786-799. Function
of the essential ATP-binding cassette protein RLI1 in translation by
promotion of pre-initiation complex assembly Dong, Hinnebusch RLI1 is
an essential yeast protein closely related in sequence to two soluble members
of the ATP-binding cassette (ABC) family of proteins that interact with
ribosomes and function in translation elongation (YEF3) or translational
control (GCN20). We found that affinity-tagged RLI1 co-purifies with eIF3,
eIF5, and eIF2, but not with other translation initiation factors, and that
RLI1 is associated with 40S ribosomal subunits in vivo. Depletion of
RLI1 in vivo leads to cessation of growth, a lower polysome content,
and decreased average polysome size. We also observed a marked reduction in
40S-bound eIF2 and eIF1, consistent with an important role for RLI1 in
assembly of 43S pre-initiation complexes in vivo. Mutations of
conserved residues in RLI1 that are expected to function in ATP hydrolysis
were lethal. A mutation in the second ABC domain of RLI1 had a
dominant-negative phenotype, decreasing the rate of translation initiation in
vivo, and the mutant protein inhibited translation of a luciferase mRNA
reporter in wild-type cell extracts. These findings are consistent with a
direct role for the ATP-binding cassettes of RLI1 in translation initiation.
RLI1-depleted cells exhibit a deficit in free 60S ribosomal subunits, and we
found RLI1-GFP in both the nucleus and cytoplasm of living cells. Thus, RLI1
may have dual functions in translation initiation and ribosome biogenesis
(Dong et al., 2004). Dong J, Lai R, Nielsen K, Hamilton C, Qiu H,
Hinnebusch AG. The essential ATP-binding cassette protein RLI1 functions in
translation by promoting preinitiation complex assembly. J Biol Chem 2004;279:42157-42168. Nuclear
surveillance and degradation of hypomodified initiator tRNAMet Krueger, Hinnebusch;
in collaboration with Anderson The
tRNA m(1)A58 methyltransferase is composed of two subunits encoded by the
essential genes TRM6 and TRM61 (formerly known as GCD10 and
GCD14), first identified as negative regulators of GCN4 translation.
We showed previously that the trm6-504 mutation results in a defective
m(1)A methyltransferase (Mtase) and a temperature-sensitive growth phenotype
that is attributable to the absence of m(1)A58 and consequential instability
of tRNAiMet. We used a genetic approach to identify the
genes responsible for initiator tRNAiMet degradation in
trm6 cells and identified three recessive extragenic mutations that suppress trm6-504
phenotypes and restore hypomodified tRNAiMet to
near-normal levels. The wild-type allele of one suppressor, DIS3/RRP44,
encodes a 3´–5´ exoribonuclease and a member of the multi-subunit
exosome complex. We have evidence that a functional nuclear exosome is
required for the degradation of tRNAiMet lacking
m(1)A58. A second suppressor gene encodes TRF4, a DNA polymerase (pol sigma)
with poly(A) polymerase activity. Whereas deletion of TRF4 leads to
stabilization of tRNAiMet, overexpression of TRF4
destabilizes the hypomodified tRNAiMet in trm6 cells.
The hypomodified, but not wild-type, pre–tRNAiMet
accumulates as a polyadenylated species whose abundance and length
distribution both increase upon Trf4p overexpression. The data indicate that
a tRNA surveillance pathway exists in yeast that requires TRF4 and the
exosome for polyadenylation and degradation of hypomodified pre- tRNAiMet. Kadaba S, Krueger A, Trice T, Krecic AM, Hinnebusch AG, Anderson
J. Nuclear surveillance and degradation of hypomodified initiator tRNAMet in
S. cerevisiae. Genes Dev 2004;18:1227-1240. Negative
regulation of GCN2 by YIH1: competition for GCN1 interaction Sattlegger, Ashcraft,
Cherkasova, Hinnebusch; in collaboration with Link We
showed previously that activation of GCN2 by uncharged tRNA is negatively
regulated in nutrient-replete medium by phosphorylation of Ser577 in GCN2 in
a manner dependent on the TOR protein kinases and TAP42, a regulator of type
2A protein phosphatases (Cherkasova and Hinnebusch, 2003). Activation of GCN2
also requires physical interaction between the GCN2-NTD and a C-terminal
segment of the GCN1 protein, most likely when both proteins are bound to
translating ribosomes. YIH1 has a domain related to the GCN2-NTD, and we
found that, when overexpressed, YIH1 competes with GCN2 for GCN1-binding and
dampens the induction of GCN4 and its target genes (Gcn- phenotype). The Gcn- phenotype associated with YIH1
overexpression is suppressed by GCN2 overexpression, and the growth defect
conferred by a constitutively active GCN2c allele is also
partially suppressed by YIH1 overexpression. In vivo, overexpressed
YIH1 binds to GCN1, reduces native GCN1/GCN2 complex formation, and
suppresses eIF2-alpha phosphorylation by GCN2. YIH1 interacts with the same
GCN1 fragment that binds to the GCN2-NTD, and the YIH1-GCN1 interaction
requires Arg-2259 in this GCN1 fragment both in vitro and in
full-length GCN1 in vivo, as found previously for GCN2-GCN1
interaction. However, deletion of YIH1 does not produce a detectable
increase in eIF2-alpha phosphorylation, suggesting that YIH1 at native levels
is not a general inhibitor of GCN2 activity. We discovered that YIH1 normally
resides in a 1:1 complex with monomeric actin, rather than with GCN1, and
that a genetic reduction in actin levels impedes the induction of GCN4.
This Gcn- phenotype was partially suppressed by
deletion of YIH1, consistent with YIH1-mediated inhibition of GCN2 in
actin-deficient cells. We suggest that YIH1 normally resides in a latent
YIH1/actin complex and is released for inhibition of GCN2 and the stimulation
of general translation under either specialized conditions or in a restricted
cellular compartment, in which YIH1 is displaced from monomeric actin
(Sattlegger et al., 2004). We have recently detected genetic
interactions between a yih1-del allele and mutations in other
actin-binding proteins, suggesting a role for YIH1 in regulating the actin
cytoskeleton. Cherkasova VA, Hinnebusch AG. Translational control by TOR and
TAP42 through dephosphorylation of eIF2alpha kinase GCN2. Genes Dev
2003;17:859-872. Sattlegger E, Swanson MJ, Ashcraft E, Jennings J, Fekete R, Link
AJ, Hinnebusch AG. YIH1 is an actin-binding protein that inhibits protein
kinase GCN2 and impairs general amino acid control when overexpressed. J
Biol Chem 2004;279:29952-29962. Requirement
of a multiplicity of co-activators for pre-initiation complex assembly;
subunit requirements for Srb mediator recruitment by GCN4 in vivo Swanson,a
Qiu, Yoon, Sumibcay,b Kim, Zhang, Hu, Hinnebusch Transcriptional
activation in eukaryotes typically involves sequence-specific DNA binding
proteins that bind upstream of promoters and recruit multi-subunit
co-activator complexes with the capacity to stimulate assembly of a
pre-initiation complex (PIC) at the promoter. Some co-activators, including
SWI/SNF and RSC, are ATP-dependent enzymes capable of remodeling the
nucleosome structure of the promoter. Other co-activators, such as the SAGA
complex, contain histone acetyltransferase (HAT) activities that facilitate
chromatin remodeling or mark promoter nucleosomes as binding sites for other
co-activators. A third class of co-activators, including the Srb and Paf1
mediators, TFIID, and CCR4-NOT, are physically associated with TATA-binding
protein (TBP), other general transcription factors (GTFs), or RNA polymerase
II (Pol II) and are thought to function as adaptors between the activator and
transcriptional machinery that promote PIC assembly. Our previous studies
showed that wild-type transcriptional activation by Gcn4p depends on multiple
co-activators, including SAGA, SWI/SNF, RSC, Srb mediator, Paf1 complex,
CCR4-NOT complex, and the MBF1 protein. We demonstrated that GCN4 can
interact in vitro with all these co-factors except Paf1 complex and
that GCN4 recruits each to its target promoters in vivo (Swanson et al.,
2003). It was not known whether these are required for assembly of the
pre-initiation complex (PIC) or for subsequent steps in the initiation or
elongation phase of transcription. We found that mutations in their subunits
reduce the recruitment of TBP and Pol II by Gcn4p at ARG1, ARG4,
and SNZ1, implicating all five co-activators in PIC assembly at Gcn4p
target genes. Recruitment of Pol II at SNZ1 and ARG1 was
eliminated by mutations in TBP or by deletion of the TATA box, indicating
that TBP binding is a prerequisite for Pol II recruitment by Gcn4p. However,
several mutations in SAGA subunits and deletion of SRB10 had a greater
impact on promoter occupancy of Pol II versus TBP, suggesting that SAGA and
Srb mediator can promote Pol II binding independently of their stimulatory
effects on TBP recruitment. The
Srb mediator is an important transcriptional co-activator for Gcn4p, as
indicated above. We have shown that three subunits of the Gal11/tail domain
of mediator, Gal11p, Pgd1p, Med2p, and the head domain subunit Srb2p make
overlapping contributions to the interaction of mediator with recombinant
Gcn4p in vitro. Each of these proteins, along with the tail subunit
Sin4p, also contributes to recruitment of mediator by Gcn4p to target
promoters in vivo. We found that Gal11p, Med2p, and Pgd1p reside in a
stable subcomplex in sin4-del cells that interacts with Gcn4p in
vitro and is recruited independently of the rest of mediator by Gcn4p in
vivo. Thus, the Gal11p/Med2p/Pgd1p triad is both necessary for
recruitment of intact mediator and appears to be sufficient for recruitment by
Gcn4p as a free subcomplex. The med2-del mutation impairs the
recruitment of TBP and Pol II to the promoter and the induction of
transcription at ARG1, demonstrating the importance of the tail domain
for activation by Gcn4p in vivo. Even though the Gal11p/Med2p/Pgd1p
triad is the only portion of Srb mediator recruited efficiently to the
promoter in the sin4-del strain, the mutant shows high-level TBP
recruitment and wild-type transcriptional induction at ARG1. Hence,
the Gal11p/Med2p/Pgd1p triad may contribute to TBP recruitment independently
of the rest of mediator. Qiu H, Hu C, Yoon S, Natarajan K, Swanson MJ, Hinnebusch AG. An
array of coactivators is required for optimal recruitment of TATA binding
protein and RNA polymerase II by promoter-bound Gcn4p. Mol Cell Biol
2004;24:4104-4117. Swanson MJ, Qiu H, Sumibcay L, Krueger A, Kim SJ, Natarajan K,
Yoon S, Hinnebusch AG. A multiplicity of coactvators is required by Gcn4p at
individual promoters in vivo. Mol Cell Biol 2003;23:2800-2820. Yoon S, Qiu H, Swanson MJ, Hinnebusch AG. Recruitment of SWI/SNF
by Gcn4p does not require Snf2p or Gcn5p but depends strongly on SWI/SNF
integrity, SRB mediator and SAGA. Mol Cell Biol 2003;23:8829-8845. Zhang F, Sumibcay L, Hinnebusch AG, Swanson MJ. A triad of subunits
from the Gal11/tail domain of Srb mediator is an in vivo target of
transcriptional activator Gcn4p. Mol Cell Biol 2004;24:6871-6886. aMark
Swanson, PhD, former Adjunct Scientist bLaarni Sumibcay, BS, former
Predoctoral Fellow COLLABORATORS James Anderson, PhD, Andrew Link, PhD,
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