Lesson 3: Translation

From RNA to protein: translation The genetic code Three possible “reading frames” THE ABC FOR THE DNA Insertion (X) or Deletion (B) THE AXB CFO RTH EDN A THE ACF ORT HED NA

Ribosomes in the cytoplasm of a eukaryotic cell

Composition of eukaryotic ribosomes

RNA-binding sites in the ribosome Each ribosome has: a binding site for mRNA three binding sites for tRNA A-site: aminoacyl-tRNA P-site: peptidyl-tRNA E-site: exit

matching amino acids to codons in mRNA tRNA molecules: matching amino acids to codons in mRNA

Translation of the genetic code: two adaptors that act one after another

mRNA translation mechanism Step1: An aminoacyl-tRNA molecule binds to the A-site on the ribosome Step2: A new peptide bond is formed Step3: The small subunit moves a distance of three nucleotides along the mRNA chain ejecting the spent tRNA molecule Step4: The next aminoacyl-tRNA molecule binds to the A-site on the ribosome Step5: . . .

The initiation phase of protein synthesis in eukaryotes Initiation complex (small ribosomal subunit + initiation factors) binds DNA and searches for start codon Large ribosomal subunit adds to the complex Translation starts . . .

Prokaryotic vs eukaryotic mRNA molecules

Structure of a typical prokaryotic mRNA molecule

5´ end capping of eukaryotic mRNA molecules

The final phase of protein synthesis binding of release factor to a stop codon terminates translation the completed polypeptide is released the ribosome dissociates into its two separate subunits

several ribosomes can simultaneously translate Polyribosomes: several ribosomes can simultaneously translate the same mRNA molecule

degradation of “unwanted” proteins in eukaryotic cells Proteasomes: degradation of “unwanted” proteins in eukaryotic cells

regulation/variation The production of a protein by a eukaryotic cell Many levels of regulation/variation

Protein import by membrane-bounded organelles ATP !!!

The role of signal sequences in protein sorting proteins destined for the ER: N-terminal signal sequence that directs them proteins destined to remain in the cytosol: no signal sequence

Free and membrane-bound ribosomes

ER signal sequence and SRP: directing ribosomes to the ER membrane The SRP binds to the exposed ER signal sequence and to the ribosome, thereby slowing protein synthesis by the ribosome. The SRP-ribosome complex then binds to an SRP receptor in the ER membrane. The SRP is then released, leaving the ribosome on the ER membrane. A protein translocation channel in the ER membrane then inserts the polypeptide chain into the membrane and starts to transfer it across the lipid bilayer.

Translocation of a soluble protein across the ER membrane A protein translocation channel binds the signal sequence and actively transfers the rest of the polypeptide across the lipid bilayer as a loop. At some point during the translocation process, the translocation channel opens sideways and releases the signal sequence into the bilayer, where it is cleaved off by an enzyme (a signal peptidase). The translocated polypeptide is released as a soluble protein into the ER lumen. The membrane-bound ribosome is omitted from this and the following two figures for clarity.

Integration of a transmembrane protein into the ER membrane An amino-terminal ER signal sequence (red) initiates transfer as in Figure 14-14. In addition, the protein also contains a second hydrophobic sequence, a stop-transfer sequence (orange). When this enters the translocation channel, the channel discharges the protein sideways into the lipid bilayer, after which the amino-terminal signal sequence is cleaved off, leaving the transmembrane protein anchored in the membrane. Protein synthesis on the cytosolic side continues to completion.

Integration of a double-pass transmembrane protein into the ER membrane An internal ER signal sequence (red) acts as a start-transfer signal and initiates the transfer of the polypeptide chain. When a stop-transfer sequence (orange) enters the translocation channel, the channel discharges both sequences sideways into the lipid bilayer. Neither the start-transfer nor the stop-transfer sequence is cleaved off, and the entire polypeptide chain remains anchored in the membrane as a double-pass transmembrane protein. Proteins that span the membrane more times contain further pairs of stop and start sequences, and the same process is repeated for each pair.

Vesicular traffic The extracellular space and each of the membrane-bounded compartments (shaded gray) communicate with one another by means of transport vesicles, as shown. In the outward secretory pathway (red arrows) protein molecules are transported from the ER, through the Golgi apparatus, to the plasma membrane or (via late endosomes) to lysosomes. In the inward endocytic pathway (green arrows) extracellular molecules are ingested in vesicles derived from the plasma membrane and are delivered to early endosomes and then (via late endosomes) to lysosomes.

Clathrin-coated pits and vesicles (A) Electron micrographs showing the sequence of events in the formation of a clathrin-coated vesicle from a clathrin-coated pit. The clathrin-coated pits and vesicles shown here are unusually large and are being formed at the plasma membrane of a hen oocyte. They are involved in taking up particles made of lipid and protein into the oocyte to form yolk. (B) Electron micrograph showing numerous clathrin-coated pits and vesicles budding from the inner surface of the plasma membrane of cultured skin cells. (A, courtesy of M.M. Perry and A.B. Gilbert, J. Cell Sci. 39:257-272, 1979, by permission of The Company of Biologists; B, from J. Heuser, J. Cell Biol. 84:560-583, 1980, by copyright permission of the Rockefeller University Press.)

mediated by clathrin-coated vesicles Selective transport: mediated by clathrin-coated vesicles Cargo receptors, with their bound cargo molecules, are captured by adaptins, which also bind clathrin molecules to the cytosolic surface of the budding vesicle. Dynamin proteins assemble around the neck of budding vesicles; once assembled, they hydrolyze their bound GTP and pinch off the vesicle. After budding is complete, the coat proteins are removed and the naked vesicle can fuse with its target membrane. Functionally similar coat proteins are found in other types of coated vesicles.

Model of transport vesicle docking Vesicles that bud from a membrane carry specific marker proteins called vesicle SNAREs (v-SNAREs) on their surface, which bind to complementary target SNAREs (t-SNAREs) on the target membrane. Many different complementary pairs of v-SNAREs and t-SNAREs are thought to play a crucial role in guiding transport vesicles to their appropriate target membranes.

Transport vesicle fusion Following the docking of a transport vesicle at its target membrane, a complex of membrane-fusion proteins assembles at the docking site and catalyzes the fusion of the vesicle with the target membrane. Fusion of the two membranes delivers the vesicle contents into the interior of the target organelle and adds the vesicle membrane to the target membrane.

Protein glycosylation in the ER Almost as soon as a polypeptide chain enters the ER lumen, it is glycosylated by addition of oligosaccharide side chains to particular asparagines in the polypeptide. Each oligosaccharide chain is transferred as an intact unit to the asparagine from a lipid called dolichol. Asparagines that are glycosylated are always present in the tripeptide sequence asparagine, X, serine or threonine, where X can be any amino acid.

The Golgi apparatus (A) Three-dimensional reconstruction of a Golgi stack. It was reconstructed from electron micrographs of the Golgi apparatus in a secretory animal cell. (B) Electron micrograph of a Golgi stack from a plant cell, where the Golgi apparatus is especially distinct. The Golgi apparatus is oriented as in (A). (C) The Golgi apparatus in a cultured fibroblast stained with a fluorescent antibody that labels the Golgi apparatus specifically. The red arrow indicates the direction of the cell's movement. Note that the Golgi apparatus is close to the nucleus and oriented toward the direction of movement. (A, redrawn from A. Rambourg and Y. Clermont, Eur. J. Cell Biol. 51:189-200, 1990; B, courtesy of George Palade; C, courtesy of John Henley and Mark McNiven.)

the regulated and constitutive pathways Exocytosis: the regulated and constitutive pathways The two pathways diverge in the trans Golgi network. Many soluble proteins are continually secreted from the cell by the constitutive secretory pathway, which operates in all cells. This pathway also supplies the plasma membrane with newly synthesized lipids and proteins. Specialized secretory cells also have a regulated exocytosis pathway, by which selected proteins in the trans Golgi network are diverted into secretory vesicles, where the proteins are concentrated and stored until an extracellular signal stimulates their secretion.

Exocytosis of secretory vesicles The electron micrograph shows the release of insulin into the extracellular space from a secretory vesicle of a pancreatic beta cell. The insulin is stored in a highly concentrated form in each secretory vesicle and is released only when the cell is signaled to secrete by an increase in glucose levels in the blood. (Courtesy of Lelio Orci, from L. Orci, J.-D. Vassali, and A. Perrelet, Sci. Am. 256:85-94, 1988.)