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STRUCTURE
Volume 13, Issue 11 , November 2005, Pages 1584-1585
The Ins and Outs of Protein Synthesis
Translation initiation and protein trafficking begin and end the process of protein synthesis. In this issue of Structure, Boehringer et al. (2005) provide the first global model of HCV IRES-80S ribosome interactions. In a second paper, Schlünzen et al. (2005) describe the structure of the bacterial 50S subunit with the ribosome binding domain of trigger factor (TF) with surprising conclusions about TF function.
Protein biosynthesis involves tight regulation of a simple chemical reaction—peptide bond formation—on a highly complex machine, the ribosome. In the past few years, structures of the ribosome have provided groundbreaking insights into how the ribosome works. However, the available structures and cryo-EM reconstructions leave many holes in our knowledge of fundamental steps in translation. For example, we are only beginning to understand the mechanisms of eukaryotic translation initiation and the structural basis of cotranslational protein folding and trafficking. In this issue of Structure, two groups present significant advances in each of these areas.
Witnessing a Hijacking. In eukaryotes, many RNA viruses hijack the translational machinery as part of their life cycle (Bushell and Sarnow, 2002). The take-over often involves cis-acting elements within the 5′-untranslated region of the viral mRNAs called Internal Ribosome Entry Sites, or IRESs. Viral IRESs direct the small (40S) ribosomal subunit to the correct start codon without the need for the full complement of initiation factors. These IRESs allow the virus to bypass numerous cellular defense mechanisms that shut down normal 5′-cap-dependent translation initiation during infection or under other stress conditions. For example, the hepatitis C virus (HCV) IRES element minimally requires only eIF3, eIF2, and eIF5B to initiate translation (Boehringer et al., 2005).
The molecular basis for HCV IRES initiation is only now starting to come into focus. Several groups have broken down the 340-nucleotide HCV IRES into subdomains and determined their structures by NMR and X-ray crystallography (Collier et al., 2002, Kieft et al., 2002, Lukavsky et al., 2003 and Lukavsky et al., 2000). These structures provide atomic resolution details of how parts of the IRES fold. Yet without the correct interacting partners—eIF3 and the 40S subunit—these subdomain structures may not represent the functional conformation of the IRES. A cryo-EM reconstruction of the HCV IRES-40S subunit complex has been determined at about 20 Å, revealing that binding of the full-length IRES leads to closing of the mRNA exit site (Spahn et al., 2001b).
Boehringer et al. (2005) have now tackled the last steps of HCV IRES initiation. The authors trapped the HCV IRES on the human 80S ribosome just at the transition from initiation to elongation by using cycloheximide, which allowed them to obtain an 20 Å cryo-EM reconstruction of the complex. Using the atomic resolution structures of the IRES subdomains and a model of the yeast 40S subunit based on a cryo-EM reconstruction (Spahn et al., 2001a), the authors built a global model for the HCV IRES bound to the ribosome (Figure 1).
Figure 1. Structures of the Ribosome in This Issue
(A) Schematic of the cryo-EM reconstruction of the HCV IRES/human 80S ribosome initiation complex. The small subunit head, platform, and binding position for RACK1 are indicated. Domain II of the IRES (PDB code 1P5P) interacts with the L1 arm on the 60S subunit. PDB coordinates used to model other domains in the IRES are indicated. Large (60S) subunit, blue; small (40S) subunit, gold; HCV IRES, green.
(B) Close-up schematic of the X-ray crystal structure of the D. radiodurans TF-BD/50S ribosomal subunit complex. The hydrophobic cleft, in gray, opens upon TF-BD binding to the 50S subunit, as indicated by arrows. A nascent polypeptide chain leaving the exit tunnel and entering the hydrophobic cleft is illustrated. Large (50S) subunit, blue; TF-BD, or “tail” of TF, salmon; head and body of TF included on the complex, red.
In the 80S ribosome/HCV IRES reconstruction, the IRES interacts with the L1 arm of the large (60S) ribosomal subunit. This observation is intriguing, as this interaction bears some similarity to that of the Cricket Paralysis Virus IRES with the L1 arm, which otherwise functions in a completely different way (Spahn et al., 2004). The L1 arm moves laterally during mRNA and tRNA translocation (Valle et al., 2003), which suggests that IRESs have commandeered the normal function of the L1 arm to facilitate clearing of the IRES after initiation.
Two aspects of the reconstruction will require future experimental investigation. First, the trapped complex lacks any bound tRNAs. This is a surprise, as one would expect to see tRNA bound to the peptidyl-tRNA site (P site) in an initiation complex. Second, the 80S-IRES complex has low levels of the regulatory protein, RACK1, bound. RACK1 binds to the head of the small ribosomal subunit and serves as a link between cellular regulatory signals and translation (Nilsson et al., 2004 and Sengupta et al., 2004). The exact role of RACK1 in signaling is not well understood, and its role in translation efficiency from the HCV IRES remains to be explored.
The Early Life of a Protein. A few seconds after translation initiates, the nascent polypeptide begins to emerge from the exit tunnel of the ribosome. This appearance is a big event for any protein and determines its ultimate fate. In bacteria, two distinct systems interact with the exit tunnel of the ribosome to direct the cotranslational folding and trafficking of proteins. The signal recognition particle (SRP) recognizes primarily nascent chains that will become integral membrane proteins (Egea et al., 2005). Nascent chains destined for the cytoplasm or for posttranslational secretion are recognized by trigger factor (TF), a multifunctional chaperone that acts synergistically with the chaperone DnaK (Deuerling et al., 1999 and Teter et al., 1999).
In the last year, structures of TF alone and the ribosome binding domain of trigger factor (TF-BD) bound to an archaeal 50S subunit provided the first structural view of how TF may assist protein folding of nascent polypeptide chains (Ferbitz et al., 2004 and Ludlam et al., 2004). Using the TF-BD/archaeal 50S subunit structure as a template, Ferbitz and coworkers proposed a model in which TF forms a hydrophobic “cradle” that covers the exit tunnel (Ferbitz et al., 2004). This molecular cradle would help protein folding by enclosing a volume roughly the size of a single protein domain.
The published structures of TF leave two issues unresolved. First, the structure of TF-BD on the archaeal 50S subunit involves a heterologous system. Archaea use a completely different chaperone to initiate protein folding, the nascent polypeptide-associated complex (NAC) (Spreter et al., 2005). Possibly as a result of the heterologous nature of the complex, only about 40 amino acids of the TF-BD could be seen in the cocrystal structure (Ferbitz et al., 2004), leaving open to question the full scope of the TF-BD/50S interaction. Second, a double deletion of TF and DnaK leads to a synthetic lethal phenotype that can be rescued by the TF-BD alone (Deuerling et al., 1999, Kramer et al., 2004 and Teter et al., 1999). The original structure provides no clear explanation for how the truncated form of TF could compensate for the double deletion.
Schlünzen et al. (2005), and independently Yonath and coworkers (Baram et al., 2005), now provide views of TF-BD bound to the bacterial 50S subunit in a homologous complex, with some surprising results. First, nearly all of the TF-BD is visible in the electron density maps of the structure presented (100 out of 112 amino acids) (Schlünzen et al., 2005). This has allowed the authors to model intact TF bound to the ribosome in a more rigorous way. Notably, interactions between TF-BD and the long loop of ribosomal protein L24 may in fact prevent a molecular cradle between TF and the ribosome from forming. Second, the fold of TF-BD changes dramatically upon 50S subunit binding, opening up a hydrophobic cleft that runs the length of the domain (Figure 1). The authors propose that this cleft, which is lined with highly conserved amino acids, may bind hydrophobic stretches of the nascent chain directly. Such an interaction would help to explain TF-BD rescue of the synthetic lethal phenotype in the double deletion of TF and DnaK (Kramer et al., 2004). The authors conclude their analysis by making the striking suggestion that TF-BD may bind signal sequences in a synergistic fashion with the SRP. There is little biochemical and structural evidence to go on (Egea et al., 2005), but the idea that SRP and TF may simultaneously interact with the same nascent chain is certainly one worth testing.
STRUCTURE
Volume 13, Issue 11 , November 2005, Pages 1584-1585
The Ins and Outs of Protein Synthesis
Translation initiation and protein trafficking begin and end the process of protein synthesis. In this issue of Structure, Boehringer et al. (2005) provide the first global model of HCV IRES-80S ribosome interactions. In a second paper, Schlünzen et al. (2005) describe the structure of the bacterial 50S subunit with the ribosome binding domain of trigger factor (TF) with surprising conclusions about TF function.
Protein biosynthesis involves tight regulation of a simple chemical reaction—peptide bond formation—on a highly complex machine, the ribosome. In the past few years, structures of the ribosome have provided groundbreaking insights into how the ribosome works. However, the available structures and cryo-EM reconstructions leave many holes in our knowledge of fundamental steps in translation. For example, we are only beginning to understand the mechanisms of eukaryotic translation initiation and the structural basis of cotranslational protein folding and trafficking. In this issue of Structure, two groups present significant advances in each of these areas.
Witnessing a Hijacking. In eukaryotes, many RNA viruses hijack the translational machinery as part of their life cycle (Bushell and Sarnow, 2002). The take-over often involves cis-acting elements within the 5′-untranslated region of the viral mRNAs called Internal Ribosome Entry Sites, or IRESs. Viral IRESs direct the small (40S) ribosomal subunit to the correct start codon without the need for the full complement of initiation factors. These IRESs allow the virus to bypass numerous cellular defense mechanisms that shut down normal 5′-cap-dependent translation initiation during infection or under other stress conditions. For example, the hepatitis C virus (HCV) IRES element minimally requires only eIF3, eIF2, and eIF5B to initiate translation (Boehringer et al., 2005).
The molecular basis for HCV IRES initiation is only now starting to come into focus. Several groups have broken down the 340-nucleotide HCV IRES into subdomains and determined their structures by NMR and X-ray crystallography (Collier et al., 2002, Kieft et al., 2002, Lukavsky et al., 2003 and Lukavsky et al., 2000). These structures provide atomic resolution details of how parts of the IRES fold. Yet without the correct interacting partners—eIF3 and the 40S subunit—these subdomain structures may not represent the functional conformation of the IRES. A cryo-EM reconstruction of the HCV IRES-40S subunit complex has been determined at about 20 Å, revealing that binding of the full-length IRES leads to closing of the mRNA exit site (Spahn et al., 2001b).
Boehringer et al. (2005) have now tackled the last steps of HCV IRES initiation. The authors trapped the HCV IRES on the human 80S ribosome just at the transition from initiation to elongation by using cycloheximide, which allowed them to obtain an 20 Å cryo-EM reconstruction of the complex. Using the atomic resolution structures of the IRES subdomains and a model of the yeast 40S subunit based on a cryo-EM reconstruction (Spahn et al., 2001a), the authors built a global model for the HCV IRES bound to the ribosome (Figure 1).
Figure 1. Structures of the Ribosome in This Issue
(A) Schematic of the cryo-EM reconstruction of the HCV IRES/human 80S ribosome initiation complex. The small subunit head, platform, and binding position for RACK1 are indicated. Domain II of the IRES (PDB code 1P5P) interacts with the L1 arm on the 60S subunit. PDB coordinates used to model other domains in the IRES are indicated. Large (60S) subunit, blue; small (40S) subunit, gold; HCV IRES, green.
(B) Close-up schematic of the X-ray crystal structure of the D. radiodurans TF-BD/50S ribosomal subunit complex. The hydrophobic cleft, in gray, opens upon TF-BD binding to the 50S subunit, as indicated by arrows. A nascent polypeptide chain leaving the exit tunnel and entering the hydrophobic cleft is illustrated. Large (50S) subunit, blue; TF-BD, or “tail” of TF, salmon; head and body of TF included on the complex, red.
In the 80S ribosome/HCV IRES reconstruction, the IRES interacts with the L1 arm of the large (60S) ribosomal subunit. This observation is intriguing, as this interaction bears some similarity to that of the Cricket Paralysis Virus IRES with the L1 arm, which otherwise functions in a completely different way (Spahn et al., 2004). The L1 arm moves laterally during mRNA and tRNA translocation (Valle et al., 2003), which suggests that IRESs have commandeered the normal function of the L1 arm to facilitate clearing of the IRES after initiation.
Two aspects of the reconstruction will require future experimental investigation. First, the trapped complex lacks any bound tRNAs. This is a surprise, as one would expect to see tRNA bound to the peptidyl-tRNA site (P site) in an initiation complex. Second, the 80S-IRES complex has low levels of the regulatory protein, RACK1, bound. RACK1 binds to the head of the small ribosomal subunit and serves as a link between cellular regulatory signals and translation (Nilsson et al., 2004 and Sengupta et al., 2004). The exact role of RACK1 in signaling is not well understood, and its role in translation efficiency from the HCV IRES remains to be explored.
The Early Life of a Protein. A few seconds after translation initiates, the nascent polypeptide begins to emerge from the exit tunnel of the ribosome. This appearance is a big event for any protein and determines its ultimate fate. In bacteria, two distinct systems interact with the exit tunnel of the ribosome to direct the cotranslational folding and trafficking of proteins. The signal recognition particle (SRP) recognizes primarily nascent chains that will become integral membrane proteins (Egea et al., 2005). Nascent chains destined for the cytoplasm or for posttranslational secretion are recognized by trigger factor (TF), a multifunctional chaperone that acts synergistically with the chaperone DnaK (Deuerling et al., 1999 and Teter et al., 1999).
In the last year, structures of TF alone and the ribosome binding domain of trigger factor (TF-BD) bound to an archaeal 50S subunit provided the first structural view of how TF may assist protein folding of nascent polypeptide chains (Ferbitz et al., 2004 and Ludlam et al., 2004). Using the TF-BD/archaeal 50S subunit structure as a template, Ferbitz and coworkers proposed a model in which TF forms a hydrophobic “cradle” that covers the exit tunnel (Ferbitz et al., 2004). This molecular cradle would help protein folding by enclosing a volume roughly the size of a single protein domain.
The published structures of TF leave two issues unresolved. First, the structure of TF-BD on the archaeal 50S subunit involves a heterologous system. Archaea use a completely different chaperone to initiate protein folding, the nascent polypeptide-associated complex (NAC) (Spreter et al., 2005). Possibly as a result of the heterologous nature of the complex, only about 40 amino acids of the TF-BD could be seen in the cocrystal structure (Ferbitz et al., 2004), leaving open to question the full scope of the TF-BD/50S interaction. Second, a double deletion of TF and DnaK leads to a synthetic lethal phenotype that can be rescued by the TF-BD alone (Deuerling et al., 1999, Kramer et al., 2004 and Teter et al., 1999). The original structure provides no clear explanation for how the truncated form of TF could compensate for the double deletion.
Schlünzen et al. (2005), and independently Yonath and coworkers (Baram et al., 2005), now provide views of TF-BD bound to the bacterial 50S subunit in a homologous complex, with some surprising results. First, nearly all of the TF-BD is visible in the electron density maps of the structure presented (100 out of 112 amino acids) (Schlünzen et al., 2005). This has allowed the authors to model intact TF bound to the ribosome in a more rigorous way. Notably, interactions between TF-BD and the long loop of ribosomal protein L24 may in fact prevent a molecular cradle between TF and the ribosome from forming. Second, the fold of TF-BD changes dramatically upon 50S subunit binding, opening up a hydrophobic cleft that runs the length of the domain (Figure 1). The authors propose that this cleft, which is lined with highly conserved amino acids, may bind hydrophobic stretches of the nascent chain directly. Such an interaction would help to explain TF-BD rescue of the synthetic lethal phenotype in the double deletion of TF and DnaK (Kramer et al., 2004). The authors conclude their analysis by making the striking suggestion that TF-BD may bind signal sequences in a synergistic fashion with the SRP. There is little biochemical and structural evidence to go on (Egea et al., 2005), but the idea that SRP and TF may simultaneously interact with the same nascent chain is certainly one worth testing.
