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L2: Protein Synthesis, Processing, and Regulation Introduction Outline Translation is the synthesis of proteins as directed by mRNA templates. • Translation of mRNA But it is only the first step in the formation of a functional protein. • Protein Folding and Processing • Regulation of Protein Function The polypeptide chain must then fold into the appropriate conformation and often undergoes various processing steps. • Protein Degradation Gene expression is regulated at the level of translation in both prokaryotic and eukaryotic cells. There are also multiple controls on the amounts and activities of intracellular proteins, which ultimately regulate all aspects of cell behavior. Structure of tRNAs tRNAs align amino acids with corresponding codons on the mRNA template. They are 70 to 80 nucleotides long and have characteristic cloverleaf structures resulting from complementary base pairing between different regions. The anticodon loop binds to the appropriate codon by complementary base pairing. Translation of mRNA Attachment of amino acids to specific tRNAs is mediated by enzymes called aminoacyl tRNA synthetases. Each of these 20 enzymes recognizes a single amino acid, as well as the correct tRNA to attach it to. The attachment occurs in two steps: 1. The amino acid is joined to AMP, forming aminoacyl AMP. 2. The amino acid is transferred to the 3′ CCA terminus of the tRNA and AMP is released. The amino acid is then aligned on the mRNA template by complementary base pairing. At the third codon position, base pairing is relaxed, allowing G to pair with U, and inosine (I) in the anticodon to pair with U, C, or A. The genetic code Three different reading frames Figure 6-50 Molecular Biology of the Cell (© Garland Science 2008) Molecular Biology of the Cell (© Garland Science 2008) 1 Nonstandard codon-anticodon base pairing (wobbling) Nonstandard codon-anticodon base pairing Molecular Biology of the Cell (© Garland Science 2008) Attachment of amino acids to tRNAs Translation of mRNA Ribosomes are named according to sedimentation rates in ultracentrifugation: 70S for bacterial and 80S for eukaryotic. Cells have many ribosomes, illustrating the importance of protein synthesis. E. coli has about 20,000; growing mammalian cells can have ten million. All ribosomes have two subunits; each subunit contains both rRNA and proteins. The subunits of eukaryotic ribosomes are larger and have more proteins than prokaryotic ribosomes. Ribosome structure Structure of 16S rRNA 2 Translation of mRNA Structure of the 50S ribosomal subunit It was thought that ribosomal proteins catalyzed protein synthesis, but later shown that rRNA has catalytic activity. Noller and colleagues in 1992 showed that the large ribosomal subunit is able to catalyze formation of peptide bonds (the peptidyl transferase reaction) even after 90% of the ribosomal proteins have been removed. Unambiguous evidence for rRNA catalysis came from high-resolution structural analysis of the 50S ribosomal subunit, in 2000. Ribosomal proteins are absent from the site of the peptidyl transferase reaction showing that rRNA was responsible for catalyzing peptide bond formation. Translation of mRNA Prokaryotic and eukaryotic mRNAs It is now thought that ribosomal proteins play a largely structural role, and the large ribosomal subunit functions as a ribozyme. This has evolutionary implications: RNAs are thought to have been the first self-replicating macromolecules. The role of rRNA in the formation of peptide bonds extends the catalytic activities of RNA beyond selfreplication to direct involvement in protein synthesis. This may provide an important link for understanding the early evolution of cells. Translation of mRNA Signals for translation initiation In both prokaryotic and eukaryotic cells, translation always initiates with methionine, usually encoded by AUG. The signals that identify initiation codons are different in prokaryotic and eukaryotic cells. Initiation codons in bacterial mRNAs are preceded by a Shine-Dalgarno sequence, that aligns the mRNA on the ribosome. Thus they can initiate translation at the 5′ end of an mRNA and at internal initiation sites of polycistronic mRNAs. Eukaryotic mRNAs are recognized by the 7methylguanosine cap at the 5′ terminus. The ribosomes then scan downstream until they encounter the initiation codon. 3 Overview of translation 3-5 aa/sec 100-200 aa in a min 30 000 aa (titin protein in muscels) in 2-3 h Bacteria: ca. 15-20 aa/sec Translation of mRNA Initiation of translation in bacteria In bacteria, initiation starts with a 30S ribosomal subunit bound to initiation factors IF1 and IF3. Then the mRNA, the initiator N-formylmethionyl (fMet) tRNA, and IF2 (bound to GTP) join the complex. IF1 and IF3 are released, a 50S subunit binds to the complex, and IF2 is released. Initiation of translation in eukaryotic cells (Part 1) Translation of mRNA In eukaryotes initiation requires at least 11 proteins, designated eIFs (eukaryotic initiation factors). The initiator methionyl tRNA is bound to eIF2, and the mRNA is brought to the complex by eIF4E. The ribosome then scans down the mRNA to the first AUG codon. This is accompanied by ATP hydrolysis. Initiation factors are then released, and the 60S subunit joins the complex. 4 Initiation of translation in eukaryotic cells (Part 2) Translation of mRNA Initiation of translation in eukaryotic cells (Part 3) Elongation stage of translation The mechanism of elongation in prokaryotic and eukaryotic cells is similar. The ribosome has three binding sites: P (peptidyl), A (aminoacyl), and E (exit) sites. The initiator methionyl tRNA is bound at the P site. The next aminoacyl tRNA binds to the A site by pairing with the second codon of the mRNA. An elongation factor (EF-Tu in prokaryotes, eEF1a in eukaryotes) complexed to GTP brings the aminoacyl tRNA to the complex. Translation of mRNA Selection of the correct aminoacyl tRNA determines the accuracy of protein synthesis. Translation of mRNA Base pairing alone can’t account for the accuracy of protein synthesis. Then translocation occurs, during which the ribosome moves three nucleotides along the mRNA, positioning the next codon in an empty A site. A “decoding center” in the small ribosomal subunit recognizes correct codon-anticodon base pairs and discriminates against mismatches. This step translocates the peptidyl tRNA from A to P, and the uncharged tRNA from P to E. Insertion of a correct aminoacyl tRNA into the A site triggers a conformational change that induces hydrolysis of GTP bound to eEF1α and release of the elongation factor. A new aminoacyl tRNA binds to the A site and induces release of the uncharged tRNA from the E site. Then the peptide bond is formed, catalyzed by the large ribosomal subunit and the now uncharged initiator tRNA is at the P site. 5 Translation of mRNA Regeneration of eEF1 α/GTP As elongation continues, the eEF1α (or EF-Tu) released from the ribosome bound to GDP must be reconverted to its GTP form. This requires a third elongation factor, eEF1βγ (EF-Ts in prokaryotes). Regulation of eEF1α by GTP binding and hydrolysis represents a common method of protein regulation. Elongation continues until a stop codon (UAA, UAG, or UGA) is translocated into the A site. Release factors recognize the signals and terminate protein synthesis. In prokaryotic cells RF1 recognizes UAA or UAG, RF2 recognizes UAA or UGA. In eukaryotic cells eRF1 recognizes all three stop codons. Termination of translation Polysomes Figure 6-76 Molecular Biology of the Cell (© Garland Science 2008) Translation of mRNA Translational regulation of ferritin Translation of ferritin (a protein that stores iron) mRNA is regulated by repressor proteins. When iron is absent, iron regulatory protein (IRP) binds to a the iron response element (IRE) in the 5′ UTR, blocking translation. Regulation of translation also plays a key role in modulating gene expression. Regulation includes translational repressor proteins and noncoding microRNAs. Global translational activity of cells is modulated in response to cell stress, nutrient availability, and growth factor stimulation. 6 Translation of mRNA Some translational repressors bind to specific sequences in the 3′ UTR. Some bind to initiation factor eIF4E, interfering with its interaction with eIF4G and inhibiting initiation of translation. Translation of mRNA RNA interference (RNAi) mediated by short double-stranded RNAs is used as an experimental tool to block gene expression at the level of translation. The RNA interference pathway inhibits translation and/or induces degradation of target mRNAs. Two types of small RNAs mediate RNA interference: Small interfering RNAs (siRNAs)—produced from doublestranded RNAs by the nuclease Dicer. MicroRNAs (miRNAs)—transcribed by RNA polymerase II, then cleaved by nucleases Drosha and Dicer. One strand is incorporated into the RNA-induced silencing complex (RISC). siRNAs or miRNAs that pair perfectly can induce cleavage of the targeted mRNA. Most miRNAs form mismatches that repress translation. Regulation of translation by miRNAs (Part 1) Regulation of translation by miRNAs (Part 2) Translation of mRNA Regulation of translation by phosphorylation of eIF2 and eIF2B Translation can also be regulated by modification of initiation factors. This results in global effects on overall translational activity rather than translation of specific mRNAs. Phosphorylation of eIF2 and eIF2B by regulatory protein kinases blocks exchange of bound GDP for GTP, inhibiting initiation of translation. Regulation of eIF4E: growth factors activate protein kinases that phosphorylate regulatory proteins (eIF4E binding proteins, or 4E-BPs). In the absence of growth factors, the nonphosphorylated 4E-BPs bind to eIF4E and inhibit translation. 7 Regulation of eIF4E Protein Folding and Processing Polypeptide chains must undergo folding and other modifications to become functional proteins. The 3-D conformations result from interactions between the side chains of the amino acids. All information for the correct conformation is provided by the amino acid sequence. Molecular Biology of the Cell (© Garland Science 2008) Current view of protein folding Protein Folding and Processing Chaperones are proteins that facilitate the folding of other proteins. They act as catalysts that facilitate assembly without becoming part of the assembled complex. They bind to and stabilize unfolded or partially folded polypeptides that are intermediates leading to the final correctly folded state. Chaperones bind to nascent polypeptide chains that are still being translated on ribosomes. The chain must be protected from aberrant folding or aggregation with other proteins until synthesis of an entire domain is complete. Chaperones also stabilize unfolded polypeptide chains during their transport into organelles. Example: partially unfolded proteins stabilized by chaperones are transported across the mitochondrial membrane. Figure 6-85 Molecular Biology of the Cell (© Garland Science 2008) Action of chaperones during translation Chaperones in the mitochondrion then facilitate subsequent folding. Action of chaperones during protein transport 8 Protein Folding and Processing Sequential actions of chaperones Many chaperones were initially identified as heat-shock proteins (Hsp)—expressed in cells that were subjected to high temperatures. Hsp stabilize and facilitate refolding of proteins that have been partially denatured. Hsp70 chaperones and chaperonins are found in both prokaryotic and eukaryotic cells. Hsp70 proteins stabilize unfolded polypeptide chains during translation by binding to short hydrophobic segments. The unfolded polypeptide is then transferred to a chaperonin, where folding takes place. Chaperonins consist of protein subunits arranged in two stacked rings to form a double-chambered structure. This isolates the protein from the cytosol and other unfolded proteins. Hsp60-family (chaperonin) of molecular chaperones Protein Folding and Processing Two enzymes act as chaperones by catalyzing protein folding: Protein disulfide isomerase (PDI) catalyzes disulfide bond formation. PDI is one of the most abundant proteins in the ER, where an oxidizing environment allows (S—S) linkages. Peptidyl prolyl isomerase catalyzes isomerization of peptide bonds that involve proline residues. Isomerization between the cis and trans configurations of prolyl-peptide bonds, could otherwise be a rate-limiting step in protein folding. Figure 6-87 Molecular Biology of the Cell (© Garland Science 2008) The role of signal sequences in membrane translocation Protein Folding and Processing Proteolysis—cleavage of the polypeptide chain removes portions such as the initiator methionine from the amino terminus. Many proteins have amino-terminal signal sequences that target the protein for transport to a specific destination. The signal sequence is inserted into a membrane channel as it emerges from the ribosome and the rest of the polypeptide chain passes through as translation proceeds. The signal sequence is then cleaved by a membrane protease (signal peptidase). Proteolytic processing also includes formation of active enzymes or hormones by cleavage of larger precursors. 9 Proteolytic processing of insulin Protein Folding and Processing Glycosylation adds carbohydrate chains to proteins to form glycoproteins. The carbohydrate moieties play important roles in protein folding in the ER, in targeting proteins for transport, and as recognition sites in cell-cell interactions. N-linked glycoproteins: the carbohydrate is attached to the N atom in the side chain of asparagine. O-linked glycoproteins: the carbohydrate is attached to the O atom in the side chain of serine or threonine. Linkage of carbohydrate side chains to glycoproteins Protein Folding and Processing Some eukaryotic proteins are modified with lipids, which often serve to anchor them to the plasma membrane. There are four types of lipid additions: 1. N-myristoylation: myristic acid (a 14-carbon fatty acid) is attached to an N-terminal glycine. These proteins are associated with the inner face of the plasma membrane. 2. Prenylation: prenyl groups are attached to sulfur atoms in the side chains of cysteine located near the C terminus. Many of these proteins are involved in control of cell growth and differentiation, including the Ras oncogene proteins, which are responsible for many human cancers. 3. Palmitoylation: palmitic acid (a 16-carbon fatty acid) is added to sulfur atoms of the side chains of internal cysteine residues 4. Glycolipids (lipids linked to oligosaccharides) are added to C-terminal carboxyl groups. Addition of a fatty acid by N-myristoylation They anchor the proteins to the external face of the plasma membrane. The glycolipids contain phosphatidylinositol, and are called glycosylphosphatidylinositol (GPI) anchors. Prenylation of a C-terminal cysteine residue 10 Palmitoylation Structure of a GPI anchor Feedback inhibition Regulation of Protein Function Regulation of protein function allows the cell to regulate not only the amounts but also the activities of its proteins. There are three general mechanisms of control of cellular proteins: • regulation by small molecules - most enzymes are controlled by changes in conformation, often as a result of binding small molecules. - end products of many biosynthetic pathways inhibit the enzymes that catalyze the first step in their synthesis. • phosphorylation - phosphorylation is a reversible covalent modification process that can activate or inhibit a wide variety of proteins in response to environmental signals. - it is catalyzed by protein kinases, which transfer phosphate groups from ATP to the hydroxyl groups of side chains of serine, threonine, or tyrosine. - phosphorylation is reversed by protein phosphatases, which catalyze hydrolysis of phosphorylated amino acids. • protein-protein interactions Protein kinases and phosphatases Regulation of Protein Function Other covalent modifications include methylation and acetylation of lysine and arginine residues, and nitrosylation—addition of NO groups to side chains of cysteine. O-linked glycosylation of proteins may also play a regulatory role. 11 Nitrosylation Protein Degradation Protein levels in cells are determined by rates of synthesis and rates of degradation. Differential rates of protein degradation are an important aspect of cell regulation. Many regulatory proteins have short half lives; this allows levels to change quickly in response to external stimuli. Faulty or damaged proteins are recognized and rapidly degraded. The ubiquitin-proteasome pathway Protein Degradation The major pathway of protein degradation in eukaryotic cells is the ubiquitin-proteasome pathway. Ubiquitin is highly conserved in all eukaryotes; it is a marker that targets proteins for rapid proteolysis. Ubiquitin is attached to the amino group of the side chain of a lysine residue, then more are added to form a chain. Polyubiquinated proteins are recognized and degraded by a large protease complex, the proteasome. Ubiquitin can have other functions: Addition of one ubiquitin to some proteins is involved in regulation of DNA repair, transcription, and endocytosis. SUMO (small ubiquitin-related modifier) and other ubiquitin-like proteins serve as markers for protein localization and regulators of protein activity. Autophagy Protein Degradation Protein degradation can also take place in lysosomes—membrane-enclosed organelles that contain digestive enzymes, including proteases. Lysosomes digest extracellular proteins taken up by endocytosis; and take part in turnover of organelles and proteins. Containment of digestive enzymes in lysosomes prevents uncontrolled degradation of the cell contents. Movement of proteins into lysosomes is accomplished by autophagy: vesicles (autophagosomes) enclose small areas of cytoplasm or organelles. The vesicles then fuse with lysosomes. Autophagy is activated in nutrient starvation, allowing cells to degrade nonessential proteins and organelles and reutilize the components. Autophagy also plays a role in many developmental processes, such as insect metamorphosis, which involve extensive tissue remodeling. 12 Summary • Translation is more complex in eukaryotes than in prokaryotes • Translation can be regulated at the level of initiation and elongation - usually through control of the binding and hydrolysis of GTP associated with initiation and elongation factors - regulation often includes translational repressor proteins and RNAi • Translation has an inbuilt proof-reading mechanism • Proteins undergo important processing steps/modifications: - folding - proteolysis (cleavage) - glycosylation - lipidification - phosphorylation/dephosphorylation - methylations/acetylations etc that all have impact on the stability, activity, regulation and function of the protein 13