TRANSCRIPTIONAL AND TRANSLATIONAL REGULATORY MECHANISMS IN NUTRIENT CONTROL OF GENE EXPRESSION

Alan G. Hinnebusch, PhD, Head, Section on Nutrient Control of Gene Expression
Hongfang Qiu, PhD, Staff Scientist
Jinsheng Dong, PhD, Senior Research Assistant
Fan Zhang, MS, Senior Research Assistant
Wen-Ling Chiu, PhD, Postdoctoral Fellow
Kamal Dev, PhD, Postdoctoral Fellow
Andres Garriz, PhD, Postdoctoral Fellow
Naseem Gaur, PhD, Postdoctoral Fellow
Daniel Ginsberg, PhD, Postdoctoral Fellow
Chhabi Govind, PhD, Postdoctoral Fellow
Iness Jedidi, PhD, Postdoctoral Fellow
Adesh Saini, PhD, Postdoctoral Fellow
Chi-Ming Wong, PhD, Postdoctoral Fellow
Yuen Nei Cheung, BS, Predoctoral Fellow
Kimberly Hofmeyer, BS, Predoctoral Fellow
Eun-Hee Park, BS, Predoctoral Fellow
Hafsa Rahman, BS, Predoctoral Fellow
Cuihua Hu, BA, Special Volunteer

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We study mechanisms of transcriptional and translational control regulating the expression of amino acid–biosynthetic genes by nutrients in the yeast Saccharomyces cerevisiae. Translation of the key transcriptional activator GCN4 is stimulated in amino acid–starved cells by a mechanism involving short open reading frames (uORFs) in the mRNA leader and phosphorylation of the translation initiation factor 2 (eIF2). Bound to GTP, the factor delivers initiator tRNAiMet to the 40S ribosome. Phosphorylation of eIF2 by the kinase GCN2 inhibits formation of the eIF2-GTP-tRNAiMet ternary complex (TC), reducing general protein synthesis but derepressing translation of GCN4 (the Gcd− phenotype). We analyze: (1) the physical and functional interactions of eIF2 with other initiation factors (eIF-1, eIF-1A, eIF-3, and eIF-5) and the 40S ribosome that promote TC recruitment and ribosomal scanning during general and GCN4-specific translation; (2) conformational changes that occur in the scanning pre-initiation complex governing stringent selection of AUG as start codon; (3) co-activators required for gene activation by GCN4 to define the molecular program for recruitment of chromatin remodeling enzymes as well as adaptor proteins that deliver TATA-binding protein, other general factors, and RNA polymerase II to the promoter; and (4) association of co-activators with transcribed coding sequences and their roles in histone eviction, processivity of elongation, and termination.

Roles of translation factors eIF1A and eIF1 in pre-initiation complex assembly

Fekete,¹ Cheung, Jivotovskaya,² Nielsen,³ Hinnebusch; in collaboration with Lorsch, Pestova

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 eIF-1, eIF-1A, and eIF-3. The 43S pre-initiation complex (PIC) thus formed interacts with mRNA in a manner stimulated by eIF4F and poly(A)-binding protein, and the resulting 48S complex scans the mRNA until the Met-tRNAiMet base-pairs with the AUG start codon. On AUG recognition, the GTPase-activating protein 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 molecular functions of eIF-1, eIF-1A, and eIF-3in the recruitment of TC and mRNA to the 40S ribosome, scanning, and AUG selection in vivo by generating Gcd− mutations in these factors and examining the consequences on the rate of general translation initiation, 43S/48S complex assembly, and GCN4 translational control. We are also dissecting the role of these factors and the eIF4 group of factors in scanning and accurate AUG selection by identifying mutations that prevent induction of GCN4 translation (Gcn− phenotype)—mutations that elevate or depress initiation at UUG codons (Sui− and Ssu− phenotypes, respectively) or cause scanning ribosomes to bypass AUG codons (leaky scanning). In parallel with these in vivo measurements, we characterize in vitro the effects of the mutations on various steps of PIC assembly and the conformational transitions linked to AUG selection by using cell-free extracts or, in collaboration with Jon Lorsch’s laboratory, fully reconstituted translation systems.

Previously, we showed that the eIF3 complex and eIF1 and eIF5 reside with TC in a multifactor complex (MFC), and we mapped interactions between these factors—interactions that stabilize the MFC. The N-terminal domain (NTD) of eIF3c/NIP1 interacts directly with eIF1 and eIF5 and indirectly with the TC via eIF5, and we showed that mutating residues in the NIP1 NTD confers a Gcd− phenotype that is suppressed by overexpressing TC, destabilizes the MFC, and reduces eIF2 binding to native PICs. Our findings provided evidence that MFC assembly enhances PIC assembly in vivo. More recently, we conducted an exhaustive test of this model by measuring the effects of depleting each factor on binding of all other MFC constituents to native PICs. Using “degron” mutants endowed with conditional expression of eIF2β, eIF3a plus eIF3b, or eIF5, we found that depletion of each factor reduced 40S binding of all MFC constituents, indicating that eIF2, eIF3, and eIF5 are interdependent for optimal 40S binding in vivo (Jivotovskaya et al., 2006).

Consistent with the MFC hypothesis, we identified three mutations in surface-exposed residues of eIF1—amino acids 9 and 12 in the unstructured N-terminal tail (NTT), 93 through 97 in the globular domain, and G107 at the extreme C-terminus—that conferred a Gcd− phenotype and, for 93–97 and G107R, impaired MFC stability and 40S binding of MFC components in vivo. Analysis of these eIF1 mutations in the reconstituted system by Lorsch and colleagues showed that the 9,12 and G107R mutations lowered the rate of TC loading on 40S subunits without decreasing 40S binding of eIF1 itself, whereas 93–97 decreased 40S binding affinity but not eIF1 function in TC recruitment (Cheung et al., 2007). We found that Gcd− point mutations (F131,F131) or complete truncation (ΔC) of the unstructured C-terminal tail (CTT) of eIF1A also reduced TC loading in the reconstituted system; interestingly, the 131,133 eliminated the ability of eIF1 to stimulate TC loading by eIF1A, indicating loss of a functional interaction between eIF1 and the eIF1A CTT in this reaction. We found that a Gcd− point mutation in the OB-fold of eIF1A (66–70) reduced TC loading indirectly by decreasing 40S binding of eIF1A itself both in vivo and in vitro, supporting the idea that eIF1A binds in the 40S A-site in a manner similar to that of the related, bacterial translation factor IF1 (Fekete et al., 2005, 2007).

Cheung YN, Maag D, Mitchell SF, Fekete CA, Algire MA, Takacs JE, Shirokikh N, Pestova T, Lorsch JR, Hinnebusch AG. Dissociation of eIF1 from the 40S ribosomal subunit is a key step in start codon selection in vivo. Genes Dev 2007;21:1217-30.

Fekete CA, Applefield DJ, Blakely SA, Shirokikh N, Pestova T, Lorsch JR, Hinnebusch AG. The eIF1A C-terminal domain promotes initiation complex assembly, scanning and AUG selection in vivo. EMBO J 2005;24:3588-601.

Fekete CA, Mitchell SF, Cherkasova VA, Applefield D, Algire MA, Maag D, Saini AK, Lorsch JR, Hinnebusch AG. N- and C-terminal residues of eIF1A have opposing effects on the fidelity of start codon selection. EMBO J 2007;26:1602-14.

Jivotovskaya AV, Valasek L, Hinnebusch AG, Nielsen KH. Eukaryotic translation initiation factor 3 (eIF3) and eIF2 can promote mRNA binding to 40S subunits independently of eIF4G in yeast. Mol Cell Biol 2006;26:1355-72.

Roles of eIF1A and eIF1 in scanning and AUG recognition

Fekete,¹ Cheung, Hinnebusch; in collaboration with Lorsch, Pestova

We discovered that the DDtruncation and 131,133 point mutations in the eIF1A C-terminal tail (CTT) and the substitution of residues 98 through 101 in the helical domain of eIF1A all increase initiation at UUG start codons (Sui− phenotype) and decrease leaky scanning of the AUG at GCN4 uORF1 in vivo—consistent with a higher probability of start codon selection versus continued scanning. In collaboration with Tatyana Pestova’s laboratory, we obtained in vitro evidence supporting this model by using a reconstituted mammalian system in which inhibition of primer extension (toe-printing) is used to map the locations of scanning PICs on specific mRNAs. Yeast eIF1A substitutes for mammalian eIF1A in this assay, and the eIF1A Sui− mutations reduce the ability of PICs to migrate from the cap and bypass an upstream GUG. We concluded that the CTT and helical domain of eIF1A promote scanning and that a slower rate of scanning in the eIF1A mutants is responsible for increased UUG initiation and decreased frequency of leaky scanning past AUG codons (Fekete et al., 2005, 2007).

By measuring the kinetics of eIF1A dissociation from reconstituted 48S PICs, Lorsch and colleagues found that eIF1A binding to the PIC is strengthened (resulting in slower dissociation and less rotational freedom of its CTT) when AUG occupies the P-site and eIF5 is present in the complexes. Given that both the DD truncation of eIF1A and the SUI5 mutation in eIF5 strengthen eIF1A binding at UUG in this assay and that both mutations increase UUG initiation in vivo (Sui- phenotypes), Lorsch and colleagues proposed that tighter binding of eIF1A characterizes the closed, scanning-arrested conformation of the initiation complex at the start codon. In collaboration with Lorsch’s group, we showed that the Sui− eIF1A-CTT mutation 131,133 behaved similarly to DDin strengthening eIF1A binding at UUG. Mutations in the unstructured NTT of eIF1A (residues 7–11 or 17–21) have the opposite effect and weaken eIF1A binding with UUG or AUG in the P-site. Remarkably, these NTT mutations suppress the Sui− phenotypes of mutations in eIF5 (SUI5) and eIF2β (SUI3-2) and increase leaky scanning of the GCN4 uORF1 AUG—all consistent with the reduced probability of start codon selection versus continued scanning. Thus, the eIF1A CTT and NTT mutations have opposing effects on start codon selection and binding of eIF1A to the PIC, indicating that the strength of eIF1A association with the PIC is an important determinant of AUG selection in vivo (Fekete et al., 2007). We proposed that tight binding of eIF1A mediated by the NTT stabilizes the closed conformation of the PIC required for start codon selection, whereas the CTT mediates the weaker association of eIF1A that promotes the open, scanning conformation of the PIC (Figure 3.1).

Figure 3.1

Several lines of evidence converged recently to indicate that eIF1 is a negative regulator of initiation at non–AUG codons and that it promotes an open conformation of the PIC conducive to scanning and restricts base-pairing of Met-tRNAiMet with non–AUG triplets, thereby restraining the ability of eIF5 to stimulate GTP hydrolysis by the TC and to block Pi release from GDP-Pi in the TC. These gatekeeper functions would be eliminated when AUG enters the P-site owing to eIF1 dissociation from its location near the P-site. In support of this model, we found that overexpressing eIF1 suppresses the increased UUG initiation in all Sui− mutants tested to date, consistent with the idea that increased eIF1 binding near the P-site favors the open-scanning conformation and prevents Pi release at UUGs. In collaboration with Lorsch’s group, we showed that Sui− eIF1 mutations D83G, Q84P, and 93–97 all decrease eIF1 affinity for 40S subunits and that both the Sui− phenotypes and impaired 40S binding of eIF1 are partially corrected by overexpressing the mutant proteins in vivo. Importantly, the 93–97 mutation elevates the rates of both eIF1 dissociation and Pi release from eIF2-GDP-Pi in reconstituted PICs. Working with Pestova’s group, we found that all three eIF1 mutations also increase selection of non–AUGs in the reconstituted mammalian system independent of GTP hydrolysis. Remarkably, an eIF1A NTT mutation that suppresses UUG initiation in Sui− mutants (Ssu− phenotype) reduces (rather than raises) the rate of eIF1 release from reconstituted initiation complexes. The results indicate that release of eIF1 from the 40S subunit is a critical step in AUG selection in vivo, allowing Pi release and the transition to a closed, scanning-arrested conformation of the PIC, which is modulated by eIF1A (Cheung et al., 2007) (Figure 3.1).

Functions of GCN5, the histone acetyltransferase in co-activator SAGA, in acetylation, eviction, and methylation of nucleosomes in transcribed coding regions

Govind, Zhang, Qiu, Hofmeyer, Hinnebusch

Transcriptional activators bind to enhancers (or in yeast to upstream activation sequences [UASs]) located upstream of promoters and recruit co-activator complexes that stimulate assembly of a 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 Mediator and TFIID, are physically associated with general transcription factors (GTFs) or RNA polymerase II (Pol II) and function as adaptors between the activator and transcriptional machinery. Some co-activators, including SAGA and Mediator, perform both histone modifications and adaptor functions.

Our previous studies showed that efficient transcriptional activation by GCN4 is dependent on the co-activators SAGA, SWI/SNF, RSC, and Mediator and that GCN4 recruits these cofactors to target promoters in vivo. We established that all four co-activators stimulate assembly of the PIC, as mutations in subunits of each one reduce the recruitment of TATA binding protein (TBP) and Pol II to promoters by GCN4. We also obtained evidence that SAGA and SWI/SNF stimulate promoter clearance or elongation (Govind et al., 2005). In pursuing this last observation, we discovered that SAGA is associated at high levels with the coding sequences of GCN4 target genes and with GAL1 during induction and that SAGA association with the ORF requires both transcription and high-level phosphorylation on serine-5 of the heptad repeats in the C-terminal domain (CTD) of Pol II subunit RPB1 by the kinase KIN28. We further showed that the HAT GCN5, most likely in SAGA, functions in transcribed coding sequences to (1) enhance nucleosome eviction from the highly transcribed GAL1 gene; (2) maintain high-level histone H3 acetylation in nucleosomes reassembled in the wake of elongating Pol II; (3) promote Pol II processivity to an extent that increases transcriptional output from an ORF of extended (8 kb) length; and (4) stimulates H3 lysine 4 trimethylation. Interestingly, GCN5 also opposes the effects of several histone deacetylase complexes that are likewise recruited by GCN4 to transcribed coding sequences, presumably to maintain the optimum level of H3 acetylation needed to prevent gene silencing (by hypoacetylation) or activation of cryptic promoters (by hyperacetylation) (Govind et al., 2007).

Govind CK, Yoon S, Qiu H, Govind S, Hinnebusch AG. Simultaneous recruitment of coactivators by Gcn4p stimulates multiple steps of transcription in vivo. Mol Cell Biol 2005;25:5626-38.

Govind CK, Zhang F, Qiu H, Hofmeyer K, Hinnebusch AG. Gcn5 promotes acetylation, eviction and methylation of nucleosomes in transcribed coding regions. Mol Cell 2007;25:31-42.

Role of yeast cap binding complex in regulating recruitment of cleavage factor IA to weak termination sites

Wong, Qiu, Hu, Dong, Hinnebusch

The nuclear cap binding complex (CBC) binds to the m7G cap on mRNAs in the nucleus and is known to stimulate assembly of spliceosome complexes co-transcriptionally on nascent transcripts. We obtained strong evidence that CBC is recruited directly to the m7G cap rather than binding first to activators bound to the UAS or to the phosphorylated CTD of Pol II subunit RPB1. More important, we discovered that CBC plays a direct role in preventing polyadenylation at weak termination sites in vivo. Similar to NPL3 with which it interacts, CBC carries out its antitermination function by impeding recruitment of subunits of cleavage factor (CF) IA at weak poly(A) addition sites. CBC does not function merely by recruiting NPL3 to the nascent transcript. We proposed that CBC bound to the cap of the nascent transcript could remain associated with elongating Pol II and help NPL3, an RNA binding protein, and could bind specifically to weak termination signals in the transcript as soon as they emerge from the Pol II active site, thus preventing their recognition by CF-IA. Alternatively, the proposed interaction of CBC with Pol II could form a closed loop that would topologically restrict cleavage by CF-IA, functioning in parallel with NPL3 to prevent polyadenylation at weak terminators (Wong et al., 2007).

Wong C-M, Qiu H, Hu C, Dong J, Hinnebusch AG. Yeast cap binding complex (CBC) impedes recruitment of cleavage factor IA to weak termination sites. Mol Cell Biol 2007;27:6520-31.

¹ Christie Fekete, PhD, former Postdoctoral Fellow
² Antonina Jivotovskaya, PhD, former Postdoctoral Fellow
³ Klaus Nielsen, PhD, former Postdoctoral Fellow

Collaborators

Jon Lorsch, PhD, The Johns Hopkins University School of Medicine, Baltimore, MD
Tatyana Pestova, PhD, DSc, SUNY Downstate Medical Center, Brooklyn, NY

For further information, contact alanh@mail.nih.gov.

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