11.5: Translation

Like any polymerization in a cell, translation occurs in three steps: initiation brings a ribosome, mRNA, and an initiator tRNA together to form an initiation complex. Elongation is the successive addition of amino acids to a growing polypeptide. Termination is signaled by sequences (one of the stop codons) in the mRNA and by protein termination factors that interrupt elongation and release a finished polypeptide.

The events of translation occur at A, P, and E sites on the ribosome (Figure 11.8).

Figure 11.8: A, P, and E sites, mostly on the large ribosomal subunit, are involved in mRNA translation.

11.5.1 Even Before Initiation—Making Aminoacyl-tRNAs

Translation is perhaps the most energy-intensive job a cell must do, starting with attaching amino acids to their tRNAs. The amino-acylation reaction is the same for all amino acids. A specific aminoacyl-tRNA synthase attaches a tRNA to (i.e., charges) an appropriate amino acid. This process of charging tRNAs has its own three steps and requires ATP (Figure 11.9).

Figure 11.9: The three steps of amino acid activation (tRNA acylation) require ATP hydrolysis.

In the first step, ATP and an appropriate amino acid bind to the aminoacyl-tRNA synthase. The ATP is hydrolyzed, releasing a pyrophosphate (PPi) and leaving an enzymeAMP–amino acid complex, which transfers the amino acid is transferred to the enzyme, releasing the AMP. Finally, the tRNA binds to the enzyme; the amino acid is transferred to the tRNA; and the intact enzyme is regenerated and released. The charged tRNA is now ready for use in translation. Several studies had already established that polypeptides are synthesized from their amino (N-) terminal end to their carboxyl (C-) terminal end. When it became possible to determine the amino-acid sequences of polypeptides, it turned out that around 40% of E. coli proteins had an N-terminal methionine. This suggested that all proteins begin with a methionine, but that the methionine was subsequently removed in a posttranslational processing step. It also turned out that, even though there is only one methionine codon, two different tRNAs for methionine could be isolated. One of the tRNAs was bound to a methionine modified by formylation, called formyl-methionine-tRNAfmet (fmet-tRNAf for short). The other was methionine-tRNAmet (met-tRNAmet for short), whose methionine was not formylated. tRNAmet and tRNAf each have an anticodon to AUG, the only codon for methionine. But they have different base sequences encoded by different tRNA genes. tRNAmet is used to insert an encoded methionine anywhere in a polypeptide. tRNAf, the initiator tRNA, is only used to start new polypeptides with formyl-methionine. In prokaryotes, the amino group of the methionine on met-tRNAf is formylated by a formylating enzyme to make the fmet-tRNAf. This enzyme does not recognize the methionine on met-tRNAmet. Methionine and formylated methionine structures are compared in Figure 11.10.

Figure 11.10: Discovery of met-tRNA and formyl-met-tRNAf, despite only one codon for methionine.

In E. coli, a formylase enzyme removes the formyl group from all N-terminal formyl methionines at some point after translation has begun. As noted, methionines themselves (and sometimes more N-terminal amino acids) are also removed from ~60% of E. coli polypeptides. While eukaryotes inherited both a tRNAmet, and a tRNAf (using only met-tRNAf during initiation), methionine on the latter is never formylated in the first place! What’s more, methionine is absent from the N-terminus of nearly all mature eukaryotic polypeptides. Apparently, early in evolution, the need for an initiator tRNA must have ensured a correct starting point for translation on an mRNA and therefore the growth of a polypeptide from one end to the other (i.e., from its N- to C-termini).

At that time, formylation of the N-terminal methionine may have served to block accidental addition of amino acids to the N-terminus of a polypeptide. Today, formylation is a kind of molecular appendix in bacteria. In eukaryotes at least, evolution has selected other features to replace actual formylation as a protector of the N-terminus of polypeptides.

11.5.2 Translation Initiation

Now that we have charged the tRNAs, we can look more closely at the three steps of translation. Understanding translation initiation began with cell fractionation of E. coli, the purification of molecular components required for cell-free (in vitro) protein synthesis, and finally, reconstitution experiments. Cellular RNA was purified and the 30S ribosomal subunit was separated from a ribosomal extract. These were then added to initiation-factor proteins purified from the bacterial cells. Reconstitution experiments revealed that when added to each other in the correct order, the separated fractions, along with the purified initiation factors, formed a stable 30S ribosomal subunit–mRNA complex. We now know that a short Shine-Dalgarno sequence in in the 5’ untranslated region (5’ UTR) of the mRNA forms H-bonds with its complementary sequence in the 16S rRNA in 30S ribosomal subunit. The Shine-Dalgarno sequence is just upstream of the initiator AUG codon. The first of 3 initiation steps is a stable binding of the small (30S) ribosomal subunit to the mRNA, requiring two initiation factors IF1 and IF3, which also bind to the 30S ribosomal subunit, shown in Figure 11.11.

Figure 11.11: E. coli translation Initiation was experimentally divided into 3 steps. In step 1 shown here, the small (30S) ribosomal subunit binds to mRNA. IF1 (initiation factor 1) enables complementary H-bonding between a region of the 16S rRNA to the Shine-Dalgarno sequence near the 5’ end of the mRNA. IF3 (initiation factor 3) prevents premature attachment of the large (50S) ribosomal subunit.

206 Translation Initiation: mRNA Associates with 30S Ribosomal Subunit

CHALLENGE

When adding mRNAs to ribosomal subunits in the presence of specific proteins, how might you tell whether mRNA had then bound to a ribosomal subunit? And how could you tell if the purified initiation factors had also bound to the subunit?

Next, with the help of GTP and another initiation factor (IF2), the initiator fmet-tRNAf recognizes and binds to the AUG start codon found in all mRNAs. Some call the resulting structure (seen in Figure 11.12) the Initiation Complex, which includes the 30S ribosomal subunit, IFs 1, 2, and 3, and the fmet-tRNAf.

Figure 11.12: In the second bacterial translation initiation step, a GTP-bound IF2 enables fmet-tRNAfmet (the first aminoacyl tRNA) to bind to the mRNA-bound 30S ribosomal subunit, making an initiation complex.

207 Initiation Complex Function

In the last initiation step, the large ribosomal subunit binds to this complex; IFs 1, 2, and 3 disassociate from the ribosome; and the initiator fmet-tRNAfmet is now in the P site. Some prefer to call the structure formed at this point the initiation complex (Figure 11.13).

Figure 11.13: In step three of translation initiation, GTP on IF2 is hydrolyzed, the three initiation factors dissociate from the 30S subunit, and the 50S subunit binds to the initiation complex.

208 Adding the Large Ribosomal Subunit

Initiation can happen multiple times on a single mRNA, forming the polyribosome, or polysome (already described in chapter 1). Each of the complexes formed above will engage in polypeptide elongation, described next.

11.5.3 Translation Elongation

Elongation is a sequence of protein factor–mediated condensation reactions and ribosome movements along an mRNA. As you will see, polypeptide elongation proceeds in three discrete stages and requires a considerable input of free energy.

11.5.3.a Translation Elongation 1

Elongation starts with the entry of the second aminoacyl tRNA (\(aa2-tRNA_{aa2}\)) into the A site of the ribosome drawn in by the codon-anticodon interaction and GTP hydrolysis. To get there, a GTP-bound Elongation Factor Tu (EFTu-GTP) binds to an incoming aminoacyl-tRNA, in this case \(aa2-tRNA_{aa2}\) (arrow 1). The resulting ternary complex enters the ribosome A site, activating its GTPase function (arrow 2), hydrolyzing the GTP and releasing the reaction products, EFTu-GDP and Pi, from the ribosome (arrow 3). To keep elongation moving along, elongation factor Ts (EFTs) catalyzes a GTP-GDP exchange on EFTu, regenerating EFTu-GTP (arrow 4). Figure 11.14 illustrates these activities of bacterial translation elongation.

Figure 11.14: Entry of a second aminoacyl-tRNA into the ribosomal A site starts when the aminoacyl-tRNA binds EFTu-GTP, a GTP-bound elongation factor (1). The aa2-tRNA2-EFTu-GTP complex enters the ribosome A site (2). The now active GTPase function of EFTu-GTP hydrolyzes itd GTP and the resulting EFTu-GDP and Pi come off the ribosome (3). Elongation factor Ts (EFTs) swaps GDP for GTP on EFTu to regenerating EFTu-GTP (4). These steps will be repeated in each elongation cycle of translation.

209 Elongation: Elongation Factors and GTP

11.5.3.b. Translation Elongation 2

Peptidyl transferase, a ribozyme component of the ribosome itself, links the incoming amino acid to a growing chain in a condensation reaction. Figure 11.15 shows the results.

Figure 11.15: In step 2 of translation elongation, a peptidyl transferase ribozyme activity of the 23S rRNA (on the 50S ribosomal subunit) catalyzes hydrolysis of fmet from the fmet-tRNA, and formation of a peptide bond (linkage) between the fmet and the second amino acid (aa2), still linked to its \(\rm tRNA_{aa2}\).

In this reaction, the fmet is transferred from the initiator tRNAf to \(\rm aa2-tRNA_{aa2}\) in the A site, forming a peptide linkage with aa2, leaving the tRNAf in the P site.

210 Elognation: A Ribozyme Catalyzes Peptide Linkage Formation

11.5.3.c Translation Elongation 3

For translation to continue, the ribosome must move along the mRNA to expose the next codon in it’s A site. This begins when GTP binds to elongation factor G (EFG) After formation of the dipeptidyl-tRNA on the ribosome, GTP-EFG binds to the 50S ribosomal subunit where it acts as a GTP-dependent translocase, hydrolyzing the GTP to power the movement (translocation) of the ribosome along an mRNA to expose a new codon on the A site and shifting the dipeptidyl-tRNA to the P site. The third tRNA (\(\rm tRNA_{aa3}\)) now binds in the A site; \(\rm tRNA_{aa2}\), now attached to a dipeptide, is in the P site. The \(\rm tRNA_{fmet}\) (tRNAf) that started in the A site, is now in the E site of the ribosome. Note the free energy cost of elongation: at 3 NTPs per cycle, translation is the most expensive polymer synthesis reaction in cells!

The tRNAf, no longer attached to an amino acid, will exit the E site as the next (third) aa-tRNA enters the empty A site, based on a specific codon-anticodon interaction (assisted by elongation factors and powered by GTP hydrolysis) to begin another cycle of elongation. Figure 11.16 (below) illustrates the movement of the ribosome along the mRNA.

Figure 11.16: Elongation continues as the ribosome moves along the mRNA. Binding of elongation factor G (GTP-EFG) to the 50S ribosomal subunit activates its translocase that then catalyzes GTP hydrolysis to power ribosome movement along the mRNA. The first (fmet) tRNA in the E site is ready to exit the ribosome, the dipeptidyl-tRNA is in the peptidyl (P) site, and the next codon is exposed in the A site. Entry of the next aminoacyl-tRNA will begin the next round of elongation, repeating the events in Figure 11.14.

211 Elongation: Translocase Moves Ribosome along mRNA

212 Adding the Third Amino Acid

213 Big Translation Energy Costs

As polypeptides elongate, they pass through a groove in the large ribosomal subunit. As they emerge, the previously noted E. coli formylase catalyzes removal of the formyl group from the now-exposed N-terminal initiation fmet of all growing polypeptides. While about 40% of E. coli polypeptides begin with a methionine, specific proteases catalyze the hydrolytic removal of the methionine, and sometimes more N-terminal amino acids) from the other 60%. All of these N-terminal modifications are examples of posttranslational processing.

214 The Fates of fMet and Met: Cases of Posttranslational Processing

11.5.4. Translation Termination

A ribosome’s translation of an mRNA ends when translocation exposes one of the three stop codons in the A site of the ribosome. These stop codons, creatively called ochre, amber, and opal (UAA, UAG, and UGA, respectively) are situated some distance from the 3’ end of an mRNA. The region between a stop codon and the end of the mRNA is called the 3’ untranslated region of the messenger RNA (3′ UTR).

Since there is no aminoacyl-tRNA with an anticodon to any of the stop codons, the ribosome actually stalls, and the translation slow-down is just long enough for a protein termination factor to enter the A site. This interaction causes the release of the new polypeptide and the disassembly of the ribosomal subunits from the mRNA. The process requires energy from yet another GTP hydrolysis. After dissociation, ribosomal subunits can be reassembled with an mRNA for another round of protein synthesis. Figure 11.17 illustrates translation termination.

Figure 11.17: Bacterial translation ends when the ribosome reaches a termination codon. Since there are no aminoacyl-tRNAs with anticodons to bind to them in the A site, a termination factor enters the A site instead, signaling the separation of mRNA, the new polypeptide, and the ribosomal subunits.

215-2 Translation Termination

We’ve seen some examples of posttranslational processing (formyl group removal in E. coli, N-terminal methionine removal from most polypeptides, etc.). Most proteins, especially in eukaryotes, undergo additional steps of posttranslational processing before becoming biologically active. We will see examples in upcoming chapters.