2

The Constructive Approach for Cell-free Translation

Takuya Ueda

2.1 Introduction

The cellular system for translating mRNA into the proper sequence of amino acids is a complex biological process. From an in vitro biochemical standpoint, the formation of a peptide bond between two amino acids is a simple dehydration reaction. However, in cells, the process of protein synthesis requires several additional protein factors and RNA molecules to ensure precise deciphering of the open reading frame (ORF) of themRNA. The ribosome, which is the name given to this complex of RNAs and protein, must also sustain the efficiency of the dehydration reaction. The small RNA subunit of the ribosome provides the scaffold that ensures accurate codon–anticodon pairing, while the large ribosomal RNA subunit contains the active site that catalyzes amino acid condensation. The ribosome possesses three translational RNA (tRNA) binding sites: the A-site binds to aminoacyl-tRNA; the P-site binds to peptidyl-tRNA; and the E-site is the exit site for deacylated tRNA [1]. Aminoacyl-tRNA synthetases, which catalyze the attachment of amino acids to their cognate tRNA, are also important components of translation, although they are not considered to be part of the ribosome [2]. Translation involves a group of components called translation factors, which fall into one of three categories: initiation factor (IF), elongation factor (EF), or release factor (RF). Orchestration of such a large and diverse set of functional components is crucial for protein synthesis to progress smoothly.

Here, we present an overview of the protein translation process in bacteria, and describe a cell-free system that we have developed for reconstituting translation in vitro.We also discuss the potential of our cell-free translation system in biotechnology applications.

2.2 The Process of Protein Synthesis

2.2.1 Polypeptide Synthesis

In bacteria, the protein translation is divided into three steps (Fig. 2.1). Translation is initiated when an mRNA enters the ribosome through a specific interaction between the Shine–Dalgarno (SD) sequence on the mRNA [3] and the SD-complementary sequence in the 16S ribosomal RNA (rRNA) of the 30S ribosomal. This is followed by recruitment and binding of the initiator fMet-tRNA to the AUG start codon, mediated by IF2, at the P-site of the ribosome. This complex is called the initiation complex [4, 5]. Prior to mRNA entry and fMet-tRNA association, the ribosome consists of dissociated 30S and 50S subunits, and the 30S subunit is complexed with initiation factors IF1 and IF3, and the ribosome recycling factor (RRF) [4–7]. Following dissociation of IF1, IF2 and IF3, the 50S subunit associates with the initiation complex. The transition from initiation to elongation occurs upon binding of an aminoacyl-tRNA to the A-site of the ribosome.

Several protein-encoding genes in the bacteria phage genome and in Grampositive bacteria have been shown to lack an SD sequence in their 5′ untranslated regions, yet are able to initiate translation at the appropriate AUG start codon [8]. These “leaderless” mRNAs are translated by an undissociated 70S ribosome, which differs from the canonical initiation process summarized above [9]. Although subunit dissociation does not occur following translation of leaderless mRNAs, the three initiation factors, IF1, IF2, and IF3, are strictly required, indicating that there are perhaps novel functions of IF1 and IF3 yet to be characterized [9].

The elongation factor EF-Tu is a GTP-binding protein, and, in its GTP-bound form, is involved in conveying aminoacylated elongator tRNAs to the A-site of the ribosome. Upon successful and stable matching of the correct codon–anticodon pair, EF-Tu-GTP is hydrolyzed to EF-Tu-GDP, and detaches from the ribosome [10]. EF-Tu-GTP is converted into GDP by the GTP exchange factor, EF-Ts. Following correct codon–anticodon matching, the amino acid carried by the tRNA in the Asite is covalently linked to the peptidyl-moiety of the amino acid carried by the tRNA in the P-site, and the tRNA in the A-site shifts to the P-site. This tRNAmovement is promoted by elongation factor EF-G and GTP hydrolysis [11]. The elongation cycle is repeated until a termination event takes place on the ribosome.

When the termination codon of the ORF lines up with the A-site in the ribosome, release factor RF1 or RF2 binds to the codon, and catalyzes hydrolysis of the peptidyl-moiety in the P-site to complete peptide synthesis [12]. RF1 or RF2 dissociation from the ribosome is mediated by a third release factor, RF3, and GTP hydrolysis [13]. Disassembly of the termination complex, which now consists of the mRNA and the terminal tRNA associated with the hydrolyzed polypeptide chain, is mediated by RRF. This is followed by subunit dissociation, and binding of IF1 and IF3 to the 30S subunit [6, 7]. The translation components are then in place to efficiently repeat the process of protein synthesis.

Fig. 2.1 Schematic representation of protein translation in bacteria. The process consists of three steps: initiation, elongation and termination.

2.2.2 Protein Maturation

The product of translation, the polypeptide chain, must undergo a maturation process, or post-translational modification, before it is considered a mature protein. The first event associated with maturation, subsequent to the polypeptide chain exiting the peptide tunnel of the 50S ribosomal subunit, is polypeptide folding [14, 15]. According to Anfinsen’s dogma, proteins have the potential to undergo selffolding, but in cells most protein folding is facilitated by specific proteins called chaperones. Three protein folding systems exist in bacteria: the DnaK/DnaJ/GrpE system, the GroEL/ES system, and the trigger factor system. The DnaK/DnaJ/GrpE and trigger factor systems are co-translational processes, while the GroEL/ES system is a post-translational folding process (Fig. 2.2) [14, 15]. A DnaK/trigger factor double deletion mutant appears to survive and can grow at low temperature [16, 17], and we have demonstrated recently that GroEL can participate in protein folding in a co-translational manner [18]. These results suggest that all three chaperone systems in bacteria are capable of mediating folding of nascent polypeptides into the correct secondary structure. However, we have very little knowledge of chaperone-dependent folding processes, owing to the difficulty in developing appropriate experimental methods for evaluating translation-coupled protein folding. In particular, the nature of the specific substrates for each of the individual chaperone systems remains unclear. To understand the process by which functional proteins with the correct conformation are synthesized, the relationship between substrate and chaperone systems in cells should be clarified.

Subsequent to protein folding, proteins may undergo various other posttranslational modifications, by such enzymes as protein kinases and glycosylases.

Fig. 2.2 Schematic of the chaperone network in E. coli. A nascent polypeptide is recognized by trigger factor as it exits the peptide tunnel of the ribosome. DnaK and DnaJ bind the growing polypeptide on the ribosome in a co-translational maturation process. In contrast, GroEL and GroES assist protein folding in a post-translational manner.

Fig. 2.3 Schematic of the membrane targeting and secretory processes in E. coli. (a) During translation, trigger factor binds to the nascent polypeptide chain. SRP then binds to the polypeptide, and trigger factor simultaneously detaches. The complex of SRP, nascent polypeptide and ribosome binds to membrane-associated SR, which is also associated with SecYEG. The polypeptide is then translocated into membrane with the help of YidC. (b) SecB binds to a nascent polypeptide and acts as chaperone to prevent aggregation. Trigger factor binds to and protects the polypeptide from SRP-binding. The SecB–polypeptide complex associates with SecA, and is directed to the SecYEG complex in the plasma membrane. SecA inserts the polypeptide into the membrane, the polypeptide is transported across the membrane, and the signal sequence is digested by a membrane-associated signal peptidase.

These types of modifications occur frequently in eukaryotic protein synthesis, and regulation of post-translational modification is crucial for the synthesis of mature, functional eukaryotic proteins.

Insertion of integral or trans-membrane proteins, or secreted proteins, into the cell membrane is another important aspect of polypeptide maturation. About 30% of the proteins encoded by the bacterial genome reside in the cellmembrane, or are secreted [19]. For membrane proteins (Fig. 2.3a), as the polypeptide is being translated on the ribosome it is protected from the binding of the chaperone protein SecB by trigger factor. The nascent polypeptide chain is then recognized by the Signal Recognition Particle (SRP, Ffh and 4.5S RNA complex in E. coli), which binds to a membrane-embedded SRP receptor, (SR, FtsY in E. coli) [20]. The nascent protein is translocated into the lipid-bilayer by a membrane-associated SecYEG complex [20]. With a secretory protein (Fig. 2.3b), the newly synthesized polypeptide is recognized by the chaperone protein SecB, which binds to and prevents aggregation of the growing polypeptide chain. The SecB–polypeptide complex associates with SecA, which directs the polypeptide into the translocation pore of the SecYEG complex. The signal sequence is cleaved by a signal peptidase on the membrane, and the protein is transported across the membrane and secreted [20]....