1. Aminoacyl-tRNA synthetase attaches amino acids to their specific tRNA molecules. The charging process (aminoacylation) produces a charged tRNA (aminoacyl-tRNA), using energy from ATP hydrolysis.
2. There are 20 different aminoacyl-tRNA synthetase enzymes, one for each amino acid. Some of these enzymes recognize tRNAs by their anticodon regions, and others by sequences elsewhere in the tRNA.
3. The amino acid and ATP bind to the specific aminoacyl-tRNA
synthetase enzyme. ATP loses two phosphates and the resulting AMP is bound to the amino acid, forming aminoacyl-AMP (Figure 6.10).
4. The tRNA binds to the enzyme, and the amino acid is transferred onto it, displacing the AMP. The aminoacyl-tRNA is released from the
enzyme.
5. The amino acid is now covalently attached by its carboxyl group to the 3’r end of the tRNA. Every tRNA has a 3’r adenine, and the amino acid is attached to the 3’r–OH or 2’r–OH of this nucleotide.(Figure 6.11).
台大農藝系 遺傳學 601 20000 Chapter 5 slide 26
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 6.10 Charging of a tRNA molecule by aminoacyl-tRNA synthetase to produce an
aminoacyl-tRNA (charged tRNA)
台大農藝系 遺傳學 601 20000 Chapter 5 slide 27
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 6.11 Molecular details of the attachment of an amino acid to a tRNA molecule
台大農藝系 遺傳學 601 20000 Chapter 5 slide 28
Initiation of Translation
Animation: Initiation of Translation
1. Protein synthesis is similar in prokaryotes and eukaryotes. Some significant differences do occur, and are noted below.
2. In both it is divided into three stages:
a. Initiation.
b. Elongation.
c. Termination.
3. Initiation of translation requires:
a. An mRNA.
b. A ribosome.
c. A specific initiator tRNA.
d. Initiation factors.
e. Mg2+ (magnesium ions).
台大農藝系 遺傳學 601 20000 Chapter 5 slide 29
4. Prokaryotic translation begins with binding of the 30S ribosomal
subunit to mRNA near the AUG codon (Figure 6.12). The 30S comes to the mRNA bound to:
a. All three initiation factors, IF1, IF2 and IF3.
b. GTP.
c. Mg2+.
5. Ribosome binding to mRNA requires more than the AUG:
a. RNase protection experiments have shown that the ribosome binds at a ribosome-binding site, where it is oriented to the correct reading frame for protein synthesis (Figure 6.13)
b. The AUG is clearly identified in these studies.
c. An additional sequence 8–12 nucleotides upstream from the AUG is commonly involved. Discovered by Shine and Dalgarno, these purine-rich sequences (e.g., AGGAGG) are complementary to the 3’r end of the 16S rRNA (Figure 6.14)
d. Complementarity between the Shine-Dalgarno sequence and the 3’r end of 16S rRNA appears to be important in ribosome binding to the mRNA
台大農藝系 遺傳學 601 20000 Chapter 5 slide 30
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 6.12 Initiation of protein synthesis in prokaryotes
台大農藝系 遺傳學 601 20000 Chapter 5 slide 31
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 6.13 Sequences of some prokaryotic ribosome-binding sites
台大農藝系 遺傳學 601 20000 Chapter 5 slide 32
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 6.14 Sequences involved in the binding of ribosomes to the mRNA in the
initiation of protein synthesis in prokaryotes
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000 Chapter 5 slide 33
6. Next, the initiator tRNA binds the AUG to which the 30S subunit is bound. AUG universally encodes methionine. Newly made proteins begin with Met, which is often subsequently removed.
a. Initiator methionine in prokaryotes is formylmethionine (fMet). It is carried by a specific tRNA (with the anticodon 5’r-CAU-3’r).
b. The tRNA first binds a methionine, and then transformylase attaches a formyl group to the methionine, making fMet-tRNA.fMET (a charged initiator tRNA).
c. Methionines at sites other than the beginning of a polypeptide are inserted by tRNA.Met (a different tRNA), which is charged by the same aminoacyl-tRNA synthetase as tRNA.fMet.
7. When Met-tRNA.fMet binds the 30S-mRNA complex, IF3 is released and the 50S ribosomal subunit binds the complex. GTP is hydrolysed, and IF1 and IF2 are relased. The result is a 70S initiation complex consisting of (Figure 6.14):
a. mRNA.
b. 70S ribosome (30S and 50S subunits) with a vacant A site.
c. fMet-tRNA in the ribosome’s P site.
台大農藝系 遺傳學 601 20000 Chapter 5 slide 34
8. The main differences in eukaryotic translation are:
a. Initiator methionine is not modified. As in prokaryotes, it is attached to a special tRNA.
b. Ribosome binding involves the 5’r cap, rather than a Shine-Dalgarno sequence.
i. Eukaryotic initiator factor (eIF-4F) is a multimer of proteins,
including the cap binding protein (CBP), binds the 5’r mRNA cap.
ii. Then the 40S subunit, complexed with initiator Met-tRNA, several eIFs and GTP, binds the cap complex, along with other eIFs.
iii. The initiator complex scans the mRNA for a Kozak sequence that includes the AUG start codon. This is usually the 1st AUG in the transcript.
iv. When the start codon is located, 40S binds, and then 60S binds, displacing the eIFs and creating the 80S initiation complex with initiator Met-tRNA in the ribosome’s P site.
c. The eukaryotic mRNA’s 3’r poly(A) tail also interacts with the 5’r cap.
Poly(A) binding protein (PABP) binds the poly(A), and also binds a protein in eIF-4F on the cap, circularizing the mRNA and stimulating translation.
台大農藝系 遺傳學 601 20000 Chapter 5 slide 35