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 Zhang, MS, Senior Research Assistant
Laxminarayana Burela, PhD, Postdoctoral Fellow
Wen-Ling Chiu, PhD, Postdoctoral Fellow
Kamal Dev, PhD, Postdoctoral Fellow
Christie Fekete, PhD, Postdoctoral Fellow
Andres Garriz, PhD, Postdoctoral Fellow
Daniel Ginsberg, PhD, Postdoctoral Fellow
Chhabi Govind, PhD, Postdoctoral Fellow
Iness Jedidi, PhD, Postdoctoral Fellow
Antonina Jivotovskaya, PhD, Postdoctoral Fellow
Soon-ja Kim, 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

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 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 translation initiation factor 2 (eIF2). Bound to GTP, eIF2 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 stimulating translation of GCN4. We are analyzing the physical and functional interactions of eIF2 with other initiation factors (eIFs -1, -1A, -3, and -5) and the 40S ribosome that promote TC recruitment, ribosomal scanning, and recognition of AUG codons during general and GCN4-specific translation. We also study the mechanism of kinase GCN2 activation by uncharged tRNA (the starvation signal) and the GCN1-GCN20 complex on translating ribosomes. Finally, to define the molecular program for recruitment of chromatin remodeling enzymes and adaptor proteins that both deliver TATA-binding protein, other general factors, and RNA polymerase II to the promoter and stimulate transcription elongation and proper termination at GCN4 target genes, we are analyzing numerous co-activators required for transcriptional activation by GCN4.

Role of the multifactor complex in preinitiation complex assembly and AUG selection

Jivotovskaya, Nielsen, ¹ 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, TC binds to the 40S ribosome with the help of eIFs 1, 1A, and 3. The 43S preinitiation 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 relative importance of eIF-1, eIF-1A, and eIF-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.

Previously, we showed that the eIF3 complex, eIF1, and eIF5 reside with TC in a multifactor complex (MFC), and we mapped interactions between these factors that stabilize MFC. The N-terminal domain (NTD) of eIF3c subunit (encoded by NIP1) interacts directly with eIF1 and eIF5 and indirectly with TC via eIF5. We showed previously that mutating two 10-amino acid clusters in the NIP1 NTD confers a Gcd- phenotype that is suppressed by overexpressing TC and decreases PIC assembly in vivo. These findings provided evidence that interactions of eIF3c/NIP1 with eIF1, eIF5, and TC enhance PIC assembly in vivo (Valasek et al., 2004). To provide a more exhaustive test of the importance of MFC formation and to evaluate the relative importance of eIF3, eIF2, and eIF5 in 43S complex assembly in vivo, we determined the effects of depleting each of these factors on the association of all other MFC constituents with native PICs. Using "degron" mutants endowed with conditional expression of eIF2%, 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. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. However, whereas depleting eIF3 impaired mRNA binding to 40S subunits, depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, eIF3 can function independently of eIF4G to promote binding of at least some mRNAs to native PICs, and it appears that eIF4G has a rate-limiting function downstream of mRNA recruitment in vivo (Jivotovskaya et al., 2006).

We have also investigated the function of the RNP1 motif in the predicted RNA recognition motif of the yeast eIF3b subunit PRT1 to determine its possible functions in 40S binding of eIF3, mRNA recruitment, or scanning. We found that mutating the motif (prt1-rnp1) impairs translation initiation in vivo and reduces 40S binding of eIF3 to native PICs. Several findings indicate that the rnp1 lesion reduces recruitment of eIF3 to the 40S subunit by the eIF3j/HCR1 subunit of eIF3: (1) rnp1 strongly impairs association of HCR1 with PRT1 without substantially disrupting the eIF3 complex; (2) rnp1 impairs 40S binding of eIF3 more than it affects 40S binding of HCR1; (3) overexpressing HCR1 reduces the Ts- phenotype and increases 40S-bound eIF3 in rnp1 cells; (4) the rnp1 Ts- phenotype is exacerbated by tif32-Δ6, which eliminates a binding determinant for HCR1 in eIF3a/TIF32; and (5) hcr1Δ impairs 40S binding of eIF3 in otherwise wild-type cells. Interestingly, rnp1 also reduces the levels of 40S-bound eIF5 and eIF1 and increases leaky scanning at the GCN4 uORF1. Thus, the PRT1 RNP1 motif coordinates the functions of HCR1 and TIF32 in 40S binding of eIF3 and is needed for optimal preinitiation complex assembly and AUG recognition in vivo (Nielsen et al., 2006).

The C-terminal domain of eIF1A functions in PIC assembly, scanning, and AUG selection

Fekete, Hinnebusch; in collaboration with Lorsch, Pestova

Previous biochemical analysis of translation initiation suggested that eIF1A promotes TC binding to the 40S subunit and subsequent ribosomal scanning, but there was no evidence that eIF1A is critical for these reactions in vivo. We hypothesized that if eIF1A is required for efficient TC recruitment, it should be possible to identify eIF1A mutations that mimic the effect of eIF2 phosphorylation and derepress GCN4 translation in gcn2 cells (Gcd- phenotype). The initiation factor eIF1A contains an OB-fold present in bacterial IF1 plus N- and C-terminal extensions not present in IF1. We found that truncating the C-terminus (ΔC) or mutating OB-fold residues (66-70) of eIF1A reduced general translation in vivo but increased GCN4 translation (Gcd- phenotype) in a manner suppressed by overexpressing TC. Consistent with our findings, both mutations diminished steady-state binding of TC, eIF5, and eIF3 to native PICs in cell extracts, and ΔC reduced the rate of TC binding to 40S subunits in vitro in reactions with purified components. The assembly defects of the OB-fold mutation can be attributed to reduced 40S binding of eIF1A, whereas ΔC impairs eIF1A function on the ribosome. A substitution in the C-terminal helix (98-101) also reduced 43S PIC assembly in vivo. Rather than producing a Gcd- phenotype, however, 98-101 impairs GCN4 derepression in a manner consistent with defective scanning by reinitiating ribosomes. Indeed, 98-101 allows formation of aberrant 48S complexes at non-AUG codons in an in vitro assay for scanning, and it increases initiation at UUG codons in vivo. Thus, the OB-fold is crucial for ribosome binding, and the C-terminal domain of eIF1A has eukaryotic-specific functions in TC recruitment and scanning (Fekete et al., 2005).

Functions of soluble ATP-binding cassette proteins in translation and ribosome biogenesis

Dong, Hinnebusch; in collaboration with Dean, Link

Most ATP-binding cassette (ABC) proteins function in membrane transport, but yeast encodes a family of soluble ABC proteins that includes translation elongation factor 3 and GCN20. We showed previously that GCN20 acts on the ribosome to stimulate activation of protein kinase GCN2 by uncharged tRNA. Subsequently, we showed that the soluble ABC protein RLI1 interacts physically with MFC, stimulates 43S PIC assembly, and enhances translation initiation in yeast cells (Dong et al., 2004). We recently assisted Michael Dean's laboratory in showing that the human RLI1 has similar functions in translation initiation (Chen et al., 2006). Independently, we discovered that the soluble ABC protein ARB1 shuttles from nucleus to cytoplasm and participates in ribosome biogenesis. We showed that depleting ARB1 leads to a deficit in 40S subunits that can be attributed to slower cleavage at the A0-, A1-, and A2-processing sites in 35S pre-rRNA and delayed processing of 20S rRNA to mature 18S rRNA. Depleting ARB1 also delays rRNA processing in the 60S biogenesis pathway. We further demonstrated that ARB1 co-sediments with 40S, 60S, and 80S/90S ribosomal species and is associated in vivo with other proteins (TIF6 and LSG1) implicated in 60S or 40S biogenesis. We propose that ARB1 functions as a mechanochemical ATPase in the 40S and 60S ribosomal biogenesis pathways (Dong et al., 2006). Together with our previous analysis of GCN20 and RLI1, it now appears that all soluble ABC proteins have functions connected with ribosomes and protein synthesis.

The SPT4 subunit of yeast elongation factor DSIF stimulates association of the Paf1 complex with elongating RNA polymerase II

Hu, Qiu, Hinnebusch

Transcriptional activators bind upstream of promoters and recruit co-activator complexes that stimulate assembly of a preinitiation 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. Still other 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 modification 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 co-factors 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 by GCN4. By showing that recruitment of SAGA, SWI/SNF, and Mediator by GCN4 to ARG1 is independent of the TATA element and PIC, we demonstrated that recruitment of these co-activators precedes PIC assembly, a conclusion supported by our kinetic analysis of co-activator binding at ARG1. Despite the simultaneous recruitment of co-activators to ARG1, we found that rapid recruitment of SWI/SNF depends on the HAT subunit of SAGA (GCN5), a non-HAT function of SAGA, as well as on Mediator function. SAGA recruitment, in turn, is strongly stimulated by Mediator and the RSC complex. We observed the same interdependencies in co-activator recruitment at the GCN4 target genes ARG4 and SNZ1. Recruitment of Mediator, by contrast, occurs independently of the other co-activators at ARG1 but requires SAGA at ARG4 and SNZ1. These findings established a program of co-activator recruitment and PIC assembly that distinguishes GCN4 from other yeast activators (Qiu et al., 2005; Govind et al., 2005).

The CYC8/TUP1 co-factor complex mediates repression of diverse genes in yeast and is recruited by DNA-binding proteins specific for the different sets of repressed genes. We identified CYC8 as a new co-activator for GCN4 and showed that it acts by promoting GCN4 binding to its target genes in vivo. TUP1 also contributes to the binding function but is less critically required. The impairment of GCN4 binding in cyc8Δ and tup1Δ cells is severe enough to reduce recruitment of SAGA, Mediator, TBP, and Pol II to GCN4 target promoters, most likely accounting for the transcriptional activation defect in both mutants. CYC8 and TUP1 are recruited by GCN4, consistent with a direct role for this complex in stimulating GCN4 binding to the promoter. Interestingly, GCN4 also stimulates binding of CYC8/TUP1 at the 3′ ends of its target genes, raising the possibility that GCN4 influences transcription elongation. Our findings reveal a novel co-activator function for CYC8/TUP1 at the level of activator binding and suggest that GCN4 enhances its own binding to the promoter by recruiting CYC8/TUP1 (Kim et al., 2005).

Figure 12.1

The Paf1 complex (Paf1C) interacts with RNA polymerase II and promotes histone methylation of transcribed coding sequences, but the mechanism of Paf1C recruitment was unknown. We have now shown that Paf1C is not recruited directly by GCN4 but rather is dependent on PIC assembly and phosphorylation of Serine 5 in the C-terminal domain (CTD) of the largest subunit of Pol II by kinase KIN28 for optimal association with ARG1 coding sequences. Importantly, we found that elongation factor SPT4 (equivalent to a subunit of human DSIF) is also required for Paf1C occupancy at GCN4-induced genes and for Paf1C association with Serine 5-phosphorylated Pol II in extracts, whereas SPT4-Pol II association is independent of Paf1C. Thus, it appears that SPT4 (or its partner in DSIF SPT5) provides a platform on Pol II for recruiting Paf1C. Deletion of SPT4 reduces trimethylation of Lys4 on histone H3, demonstrating a new role for yeast DSIF in promoting this Paf1C-dependent function in elongation (Qiu et al., 2006).

¹ Klaus Nielsen, PhD, former Postdoctoral Fellow

COLLABORATORS

Michael Dean, PhD, Laboratory of Genomic Diversity, NCI, Frederick, MD
Andrew Link, PhD, Vanderbilt University, Nashville, TN
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 ahinnebusch@nih.gov.