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 Ginsburg, PhD, Postdoctoral Fellow
Chhabi Govind, PhD, Postdoctoral Fellow
Iness Jedidi, PhD, Postdoctoral Fellow
Eun-Hee Park, PhD, Postdoctoral Fellow
Adesh Saini, PhD, Postdoctoral Fellow
Muhammed Rehman, Visiting Fellow
Yuen Nei Cheung, BS, Predoctoral Fellow
Cuihua Hu, BA, Special Volunteer

We study transcriptional and translational mechanisms that control the expression of amino acid– biosynthetic genes by nutrients in the yeast Saccharomyces cerevisiae. We analyze the physical and functional interactions of the of translation initiation factor eIF2 with other initiation factors (eIF-1, eIF -1A, eIF-3, and eIF-5) and the 40S ribosome that promote the eIF2-GTP-tRNA_i^Met ternary complex (TC) recruitment and ribosomal scanning during general translation and translation that is specific for the transcriptional activator GCN 4. We also study the conformational changes that both occur in the scanning pre-initiation complex and govern stringent selection of AUG as start codon. Regarding transcriptional control, we analyze co-activators required for gene activation by GCN4 to define the molecular program for recruitment of chromatin-remodeling enzymes and adaptor proteins that deliver TATA-binding protein, other general factors, and RNA polymerase II to the promoter. We also analyze association of co-activators with transcribed coding sequences and their roles in histone eviction, transcription elongation, and mRNA export from the nucleus.

Genetic identification of yeast 18S rRNA residues required for recruitment f initiator tRNA^Met and AUG selection

Dong, Rahman,¹ Pruitt,² Wong,³ Hinnebusch; in collaboration with Nanda, Lorsch, Shin

Translation of the key transcriptional activator GCN4 is stimulated in amino acid–starved cells by a mechanism involving short open reading frames (uORF) in the mRNA leader and by phosphorylation of translation initiation factor 2 (eIF2), which, bound to GTP, delivers initiator tRNA_i^Met to the 40S ribosome. Phosphorylation of eIF2 by the kinase GCN 2 inhibits formation of the eIF2-GTP-tRNA_i^Met ternary complex (TC), reducing general protein synthesis but derepressing translation of GCN4, with attendant transcriptional activation of amino acid biosynthetic–genes (the Gcd⁻ phenotype).

In translation initiation, the AUG start codon is decoded in the peptidyl (P) site of the ribosome by initiator Met-tRNA_i^Met. However, given the near absence of information on the importance of particular residues in 18S rRNA for efficient P-site binding of tRNAi Met and AUG selection during initiation in eukaryotes, researchers are illuminating the ribosomal determinants of tRNA binding to the P-site during elongation in bacteria using high-resolution crystal structures of 70S ribosomes bound to mRNA with cognate tRNA in the P-site. While crystal structures of eukaryotic ribosomes do not exist, the three-dimensional structure of the eukaryotic 80S ribosome appears to be similar to that of bacterial 70S ribosomes. Hence, it is possible to make good predictions from the bacterial crystal structures about the locations of specific residues in yeast 18S rRNA that reside in the conserved core of the 40S subunit. Translation of GCN4 mRNA is a sensitive indicator of the recruitment of eIF2•GTP•Met-tRNAi Met TC to 40S subunits in yeast cells, a property that we have exploited to identify mutations in eIFs that reduce TC assembly or impair TC recruitment and permit constitutive derepression of GCN4 translation (Gcd⁻ phenotype). We used the Gcd⁻ selection to obtain the first functional evidence for specific residues in 18S rRNA required for efficient TC binding and AUG selection by 40S subunits in vivo. By random mutagenesis of rDNA, we obtained mutations in the residue corresponding to bacterial A928 in conserved helix 28 (h28) that appear to derepress GCN4 translation by reducing the rate of TC loading on post-termination 40S subunits scanning downstream from uORF1. Interestingly, given that such mutations greatly increase the bypass, or “leaky scanning,” of AUG codons at uORFs during primary initiation events, it is possible to distinguish these mutations from existing Gcd⁻ mutations in various eIFs. The A928 substitutions could allow TC to dissociate from the PIC or disturb the orientation of Met-tRNA_i^Met in the P-site in a way that allows AUG to be ignored during the scanning process. Indeed, our biochemical studies reveal defects in both the association rate and stability of TC binding to 40S subunits conferred by the nonlethal A928U substitution (Dong et al., 2008).

Figure 2.1
Substituting nucleotides predicted to contact initiator tRNA in the 40S ribosomal P-site derepress translation of GCN4 mRNA in yeast cells.

The A928:U1389 base pair in helix 28 is not conserved in bacteria and was not previously implicated in P-site binding of tRNA; however, h28 contains a “bulge” residue, G926, that directly contacts the P-site codon in the crystal structures of bacterial 70S elongation complexes. We mutated G926 and other residues that contact the P-site tRNA anticodon (C1400) or ASL (G1338, A1339) in the bacterial 70S crystal structures and found that many such mutations also confer Gcd⁻ phenotypes and increased leaky scanning. Together, our results implicate a novel residue of h28 and a subset of residues making direct contacts with P-site tRNA in bacterial 70S elongation complexes in the stable anchoring of Met-tRNAi Met to the P-site and efficient AUG recognition during scanning by eukaryotic PICs (Figure 2.1) (Dong et al., 2008).

Dong J, Nanda JS, Rahman H, Pruitt MR, Shin BS, Wong CM, Lorsch JR, Hinnebusch AG. Genetic identification of yeast 18S rRNA residues required forefficient recruitment of initiator tRNA^Met and AUG selection. Genes Dev 2008;22:2242-2255.
Sonenberg N, Hinnebusch AG. New modes of translational control in development, behavior and disease. Mol Cell 2008;28:721-729.

Ribosomal protein L33 is required for ribosome biogenesis, subunit joining, and repression of GCN4 translation

Hinnebusch; in collaboration with Martín-Marcos, Tamame

We identified a new complementation group of Gcd⁻ mutants defined by the gcd17-1 mutation, which we showed to be an allele of RPL33A, one of the two genes encoding the essential 60S ribosomal subunit protein L33. The gcd17-1 mutation, which substitutes Gly-76 with Arg in rpL33A (a-G76R), impedes accumulation of 60S ribosomal subunits at the restrictive growth temperature. The impeded accumulation engenders halfmer polysomes, indicating a decreased rate of 60S–40S subunit joining at the final step of initiation and a strong reduction in general protein synthesis. Interestingly, at permissive temperature, a-G76R derepresses GCN4 translation and produces a Gcd⁻ phenotype stronger than that produced by deleting RPL33A (ΔA). This outcome is remarkable because a-G76R is less severe than ΔA in reducing 60S subunits at the permissive temperature. Analysis of GCN4-lacZ reporters suggests that both a-G76R and ΔA mutations derepress GCN4 translation in the presence of high TC levels (i.e., in the gcn2 background) by impairing 60S–40S subunit joining at uORF4 of the GCN4 mRNA leader. Thus, a-G76R might impair an important intersubunit bridge required for efficient 80S initiation complex formation. We propose that inefficient subunit joining at uORF4 allows 40S subunits to abort initiation at the uORF4 start site, to resume scanning, and to reinitiate downstream at GCN4. Our finding that the Gcd⁻ phenotype of the a-G76R mutation is suppressed by overexpressing tRNA_i^Met further suggests that dissociation of tRNA_i^Met from the 40S subunit is responsible for the postulated abortive initiation events at uORF4. Our data indicate that rpL33 plays a critical role in the ribosome biogenesis pathway required for efficient production of ribosomal subunits and a second role in 40S–60S subunit joining. Both activities contribute to repression of GCN4 translation under nonstarvation conditions and, hence, proper functioning of the general amino acid control (Martín-Marcos et al., 2007).

Martín-Marcos P, Hinnebusch AG, Tamame M. Ribosomal protein L33 is requiredfor ribosome biogenesis, subunit joining and repression of GCN4 translation. Mol Cell Biol 2007;27:5968-5985.

eIF3a cooperates with sequences 5′ of uORF1 to promote resumption of scanning by posttermination ribosomes for re-initiation on GCN4 mRNA

Hinnebusch; in collaboration with Szamecz, Valášek

A crucial but vaguely understood feature of GCN4 translational control is the highly disparate capacities of uORF1 and uORF4 to permit efficient resumption of scanning after translation termination. AU-rich sequences surrounding the stop codon of uORF1 favor resumption of scanning and re-initiation, whereas GC-rich sequences flanking the uORF4 stop codon likely trigger ribosome release. We demonstrated that sequences 5′ of uORF1 are also critical for re-initiation. However, little was known about the trans-acting factors crucial for the retention of post-termination 40S subunits at the uORF1 stop codon and for the resumption of scanning required for re-initiation. We previously found that deleting the N-terminal domain (NTD) and C-terminal domain (CTD) of the a/TIF32 subunit of eIF3 impaired association of otherwise intact eIF3 complexes with the 40S and that the NTD can interact with the 40S protein RPS0A. Here, we found that a partial deletion of the RPS0A-binding site in the tif32-NTD (Δ8) is not lethal but reduces the amount of 40S-bound eIFs in vivo, consistent with the idea that the eIF3a-NTD forms a crucial bridge between eIF3 and the 40S. Strikingly, the Δ8 truncation provokes a severe Gcn⁻ phenotype with the novel characteristic of impairing retention of post-termination 40S ribosomes at the uORF1 stop codon. Genetic epistasis interactions between mutations in stimulatory sequences upstream of uORF1 and Δ8 indicate that eIF3a interacts with the re-initiation–enhancing element located 5′ of uORF1. Thus, we propose that interaction between eIF3a-NTD and sequences 5′ of uORF1 at or near the mRNA exit channel of the 40S subunit stabilizes its association with mRNA and promotes the resumption of scanning for downstream re-initiation (Szamecz et al., 2008).

Szamecz B, Rutkai E, Cuchalová L, Munzarová V, Herrmannová A, Nielsen KH, Burela L, Hinnebusch AG, Valášek L. eIF3a cooperates with sequences 5′ of uORF1 to promote resumption of scanning by posttermination ribosomes for reinitiation on GCN4 mRNA. Genes Dev 2008; 22:2414-2425.

SUS1 is recruited to coding regions and functions during transcription elongation in association with SAGA and TREX2

Govind, Hinnebusch; in collaboration with Pascual García, Rodríguez-Navarro

Gene expression in eukaryotes depends on the coordinated action of multiprotein complexes that regulate transcription, mRNA biogenesis, or export of mature mRNA from the nucleus. The identification of SUS1 revealed an important link between transcription and mRNA export. SUS1 interacts with the transcriptional co-activator SAGA and the nuclear pore–associated complex TREX2, which is composed of SAC3, THP1, and CDC31. SUS1, SAC3, and THP1 mediate the post-transcriptional tethering of active genes to the nuclear rim for transcription-coupled mRNA export. In addition to the HAT (histone acetyl transferase) subunit GCN5, SAGA contains the ubiquitin protease UBP8, which, together with SUS1 and SGF11, forms a module in SAGA for deubiquitinylation of H2B. Given that binding of SUS1 to SAGA depends on UBP8 and SGF11, the deubiquitinylation module works at the junction between SAGA-dependent transcription and nuclear mRNA export. We showed previously that SAGA localizes to transcribed coding sequences and that GCN5-dependent acetylation promotes nucleosome eviction and processivity of RNA polymerase II (RNAP II) during elongation (Govind et al., 2007). The association of SAGA with coding sequences is dependent on phosphorylation of the CTD of the RNAP II subunit RPB1, indicating that SAGA could interact with actively transcribing RNAP II during elongation.

We have investigated the mechanisms underlying SUS1’s role in coupling transcription and mRNA export. We found that SUS1 is required for gene transcription in a length-dependent fashion, indicating an active role in mRNA biogenesis during transcription elongation. Consistently, SUS1 co-purifies with the elongating form of RNAP II phosphorylated on Ser5 and Ser2 of the CTD, the mRNA adaptor YRA1, and the export factor MEX67. We also provide evidence that SUS1 is associated at high levels with ARG1 coding sequences and that SUS1 occupancy of the ORF requires transcription and Ser5 CTD phosphorylation. Loss of the SAGA subunit SGF73 prevents SUS1 binding to SAGA and, unexpectedly, partially disrupts SUS1-TREX2 association. Whereas UBP8 and SAC3 both promote association of SUS1 with the ARG1 UAS and coding region, SGF73 is more critical for these interactions. Our data suggest a mechanism by which SUS1 plays a pivotal role in transcription during elongation mediated by both SAGA and TREX2 complexes (Pascual-García et al., 2008).

Govind CK, Zhang F, Qiu H, Hofmeyer K, Hinnebusch AG. Gcn5 promotesacetylation, eviction and methylation of nucleosomes in transcribed codingregions. Mol Cell 2007;25:31-42.
Pascual-García P, Govind CK, Queralt E, Cuenca-Bono B, Llopis A, Chavez S, Hinnebusch AG, Rodríguez- Navarro S. Sus1 is recruited to coding regions and functions during transcription elongation in association with SAGA and TREX2. Genes Dev 2008;22:2811-2822.

Disrupting vesicular trafficking at the endosome attenuates transcriptional activation by GCN4

Zhang, Gaur, Kim,⁴ Qiu, Swanson,⁵ Hinnebusch; in collaboration with Hašek

We and others have previously identified numerous mutants defective in factors required for translational induction of GCN4 mRNA or lacking co-activators required for transcriptional activation by GCN 4 on the basis of their sensitivity to sulfometuron methyl (SM ) or other inhibitors of amino acid biosynthesis. To identify novel factors involved in general amino acid control, we screened a library of haploid deletion mutants for SM sensitivity (SM). Surprisingly, we identified several SM^S/Gcn⁻ strains with deletions of genes involved in vesicular protein trafficking at the late endosome/multivesicular body (MVB). The late endosome/MVB plays a key role in coordinating vesicular transport of proteins among Golgi, vacuole/lysosome, and plasma membrane. The vps mutants are defective for an array of molecules required for producing vesicles with the appropriate cargo proteins or for the tethering and fusion of vesicles at the correct target membranes. We found that GCN4 function is impaired to the greatest extent in class C and D vps mutants, which are defective for various aspects of vesicle fusion at the endosome. Mutations in these factors impair activation of GCN4 target promoters and reduce pre-initiation complex (PIC) assembly at ARG1 without reducing the amount of GCN4 bound to the UAS_GCRE in vivo. We also observed SM^S phenotypes for class E vps mutants, which lack factors needed to sort cargo proteins into intralumenal vesicles (ILV) at the MVB for subsequent transport to the vacuole lumen. The heteromeric protein complexes ESCRT-I, ESCRT-II, and ESCRT-III, which bind ubiquitinated cargo to the MVB outer membrane, carry out the sorting function. The AAA-ATPase VPS4 then recycles E-III and segregates the cargo into ILVs. Class E vps mutants accumulate cargo proteins in aberrant structures lacking ILVs, called class E compartments. The missorted proteins include vacuolar hydrolyases, which are improperly matured and capable of proteolyzing other cargoes that accumulate in the class E compartment. Our detailed analysis of two class E mutants lacking a key component of ESCRT complex E-II (snf8Δ) or E-III (snf7Δ) revealed significant reductions in activation by nuclear-localized GCN4.

Interestingly, we found class E mutant vps4Δ, which is unable to generate IL Vs or transport MVB cargo correctly to the vacuole, to have a considerably weaker Gcn⁻ phenotype than does the snf7Δ mutant lacking an E-III subunit. Moreover, the stronger Gcn⁻ phenotype of snf7Δ cells was diminished by deletion of vacuolar protease PrA (pep4Δ). These genetic findings suggest that transcriptional attenuation in snf7Δ cells results, at least partly, from PrA-dependent proteolysis of cargo proteins in the class E compartment rather than from the inability to transport cargo via ILVs to the vacuole. Combining these results with our finding that defects in endocytosis alone do not confer strong Gcn⁻ phenotypes, we propose that either impaired delivery of MVB cargo originating in the Golgi (class C/D mutants) or rapid proteolysis of the cargo in the aberrant class E compartment (snf7Δ and snf8Δ mutants) is a key condition of MVB dysfunction that leads to a strong reduction in transcriptional activation by GCN4.

¹Hafsa Rahman, BS, former Predoctoral Fellow
²Margaret R. Pruitt, former Summer Student
³Chi-Ming Wong, PhD, former Postdoctoral Fellow
⁴Soon-ja Kim, PhD, former Postdoctoral Fellow
⁵Mark Swanson, Ph.D, former Postdoctoral Fellow

Collaborators

Jiří Hašek, PhD, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Jon Lorsch, PhD, The Johns Hopkins University School of Medicine, Baltimore, MD
Pilar Martín Marcos, PhD, Instituto de Microbiología Bioquímica, CSIC/Universidad de Salamanca, Salamanca, Spain
Jagpreet Nanda, PhD, The Johns Hopkins University School of Medicine, Baltimore, MD
Pau Pascual García, PhD, Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain
Susana Rodríguez-Navarro, PhD, Centro de Investigación Príncipe Felipe (CIPF), Valencia, Spain
Byung-Sik Shin, PhD, Program in Cellular Regulation and Metabolism, NICHD, Bethesda, MD
Béla Szamecz, PhD, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Mercedes Tamame, PhD, Instituto de Microbiología Bioquímica, CSIC/Universidad de Salamanca, Salamanca, Spain
Leos Valášek, PhD, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

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