MCB 201 Gene Expression - Spring Semester 2004
Lecture 7 (Stepwise Synthesis of Proteins on Ribosomes)
Section 4.5
1. The step in gene expression known as translation, or protein synthesis, is complex and can be conveniently divided into three stages: initiation, elongation and termination.
Initiation:
2. Figure 4-35, Lodish4e: Two types of methionine tRNA are found in all cells. It is crucial that protein synthesis start at exactly the right codon to read the genetic code in mRNA in the correct reading frame. The most frequently used start codon in both prokaryotic and eukaryotic cells is AUG, which encodes the amino acid methionine. What makes the start different from the addition of a methione internally in the polypeptide chain is that a special tRNA, used just to read the start codon, is present, tRNAi. When this tRNAi is charged with Met to form Met-tRNAi, this compound binds into the P site of ribosomes. The P site always holds the tRNA to which the growing polypeptide chain is attached. In contrast, the second type of Met-tRNA binds only in the A site of the ribosome. This site always holds the next amino acid to be added to the growing polypeptide chain. The consequences of these different sites will become clear soon. Bacterial initiation has the added twist that the initiator methionine is formylated, and so we have fMet-tRNAi. The vertebrate immune system actually keys on the presence of fMet at the N-terminus of most bacterial proteins and not on its own proteins to stimulate an inflammatory response when the presence of fMetXXXX- is detected in tissues. Inflammation, painful though it may be, is often the first major tissue-level defense response mounted against a bacterial pathogen.
3. Figure 4-25, Lodish5e: Eukaryotic initiation of protein synthesis. A crucial step is the formation of the so-called 'ternary complex' (ternary=three). This is composed of eIF2-GTP bound to Met-tRNAi. The ternary complex binds to the eukaryotic 40S (small) ribosomal subunit to which is bound two additional initiation factors that stabilize the ternary complex. An important gene expression control point is the molecule eIF2B, which can be phosphorylated on a serine residue, after which it cannot bind to Met-tRNAi, effectively inhibiting protein synthesis. The complex, now called the 43S preinitiation complex, binds to the 5' cap of the mRNA with the help of yet another initiation factor, eIF4. This one includes a helicase activity that removes RNA secondary structure and allows the ribosome complex to move or scan along the mRNA until an AUG start codon is found. The selection of the correct start site is aided by the presence of surrounding sequence information, called the Kozak sequence, again after their discoverer). Once the start site is located, the binding of the 60S subunit occurs to complete the 80S initiation complex.
Elongation:
4. Figure 4-26, Lodish5e: The elongation cycle in protein synthesis visualized for E. coli ribosomes. This figure emphasizes the shapes of the ribosomes and the approximate positions of the bound tRNAs. The key steps in elongation are: 1) entry of each aa-tRNA into the A site of the ribosome, 2) peptide bond formation, and 3) translocation or movement of the ribosome to position over the next codon in the mRNA. The process has its own set of elongation factors called EFs.
One of the most interesting recent findings is that the essential process of making the next peptide bond, the peptidyltransferase reaction is carried out not by ribosomal proteins, as initially thought, but by 23S rRNA in the large ribosomal subunit. This was convincingly shown when large subunits were stripped of most of their proteins and found to still carry out the reaction. Other studies, in which a segment of purified 23S rRNA was mixed with molecules similar to aa-tRNA and peptidyl-tRNA, showed that the peptidyltransferase reaction required only these components.
Media Connections: Focus animation: Protein synthesis
5. Figure 4-27, Lodish5e: Overall structure of the E. coli ribosome at 25A resolution inferred from cryoelectron microscopy and three-dimensional reconstruction based on the analysis of 4300 individual projections. The diagram shown here of a ribosome translating an mRNA and producing a polypeptide chain is the summation of years of effort to understand this fundamental process. Note the position of the mRNA near the anticodon loop of tRNA and the position of the polypeptide, which is actually in a channel through the large subunit that begins within 10-15 Angstroms of the 3' amino-acylated acceptor stem of the tRNAs bound in the ribosome. Note the three different positions in the ribosome occupied by three different tRNAs, the A (acceptor), P (polypeptide) and E (exit) positions will be discussed in detail later.
6. Figure 4-28, Lodish5e: Structure of the T. thermophilus 70S ribosome as determined by x-ray crystallography. Panel A shows the same side view diagrammed in Figure 4-26. The lare 50S subunit is on top and the smaller 30S subunit is on the bottom. The ribosomal proteins are located mainly on the outside and the ribosomal RNA on the inside of the subunits. The tRNA are docked at the A site (blue), P site (yellow) and E site (green). They are visible at the interface of the two subunits with their anticodon loops pointing into the small ribosomal subunit. Panel B: view of the large ribosomal subunit rotated 90 degrees about the horizontal axis, exposing the face that interacts with the small subunit. The tRNA anticodon loops point out of the page. The anticodons of the tRNAs in the A and P sites interact with mRNA codons in the small subunit. Panel C: View of the face of the small subunit that interacts with the large subunit. The tRNA anticodon loops point into the page. An important region for us to understand is shown in this panel. The acceptor stems of tRNAs in the A and P sites are close together so that the amino group of the acylated tRNA in the A site can react with the carboxyl-terminal C of the peptidyl-tRNA in the P site. This is the peptidyltransferase active site located in the large subunit of the entact 70S ribosome.
Termination:
7. Figure 4-29, Lodish5e: Termination of translation. When the translating ribosomes encounter a stop codon (UAA, UAG, UGA), there are no aa-tRNA that recognize these codons; however, there are proteins called release factors (RF) that can bind into the acceptor site of the ribosome. Two of these RF1 and RF2 have a similar shape to tRNA, an example of molecular mimicry. A third RF that carries a bound GTP (RF3-GTP) enters the complex and provides the energy for release of the finished polypeptide chain.
8. Figure 4-30, lodish5e: Eukaryotic mRNA forms a circular structure owing to interactions of three proteins. This is a force-field micrograph showing circular mRNA that forms by the binding of purified poly(A)-binding protein I and the eukaryotic initiation factors eIF4E and eIF4G. The 5' and 3' ends of the mRNA in these structures is held together by protein-protein and protein-mRNA interactions. This is shown in the diagram on the next slide.
9. Figure 4-31, Lodish5e: Formation of circular eukaryotic mRNA by protein-protein interactions bridging the 5' and 3' ends. Force-field electron microscopy has been used to demonstrate the ability of eukaryotic mRNA to form circles. There is experimental evidence that these circles are held together with proteins. The poly(A)binding protein (PABI) at the 3' end of the mRNA interacts with a complex of proteins called eIF4 at the 5' cap of mRNA. Model of protein synthesis on circular polysomes and recycling of ribosomal subunits. What is the functional value of forming mRNA circles. One hypothesis is shown here. When the release step occurs in translation, the ribosomes dissociate into small and large subunits which along with initiation factors are reused by the cell to make another initiation complex. Since this complex usually forms on the 5' end of the mRNA, having the 3' end with the stop site nearby would keep the released components in the same vicinity as the new start site from the next round of translation.
Media Connections: Overview Animation: Life Cycle of an mRNA.
Return to Lecture Index Page
