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Carboxylterminal residues are also sometimes modified.Loss of Signal Sequences As we shall see, the 15 to 30 residues at theamino-terminal end of some proteins play a role in directing the protein to its ultimate destination in the cell. Such signal sequences areultimately removed by specific peptidases.925Figure 26—31 The coupling of transcription andtranslation in bacteria.
The mRNA is translated byribosomes while it is still being transcribed fromDNA by RNA polymerase. This is possible becausethe mRNA in bacteria does not have to be transported from a nucleus to the cytoplasm before encountering ribosomes. In this schematic diagramthe ribosomes are depicted as smaller than theRNA polymerase. In reality the ribosomes (Mr2.5 x 106) are an order of magnitude larger thanthe RNA polymerase (Mr 3.9 x 105).Part IV Information Pathwayscoo"H 3 N-C—HOHSN—C—HFigure 26-32 Some modified amino acid residues,(a) Phosphorylated amino acids, (b) A carboxylatedamino acid, (c) Some methylated amino acids.CH2—O—P—O"O"PhosphoserineCOO"+ IH3N—C—H OIIII|H-C-O-P-O"CH30"PhosphothreoninePhosphotyrosine(a)COO"H3N—C—HCH2CH"OOCy-Carboxyglutamate(b)COO"I+COO"I+H3N—C—HH3N—C—HCH2CH2CH2CH2CH2CH2CH2CH2++NH 2CH3 CH3CH3DimethyllysineMethyllysineCOO"H3N-C-H+COO"IH3N— C—HCH2+NHCH2CH 2CH2CH 2CH2C=0NOCH3ITrimethyllysineMethylglutamate(c)Modification of Individual Amino Acids The hydroxyl groups of certain Ser, Thr, and Tyr residues of some proteins are enzymaticallyphosphorylated by ATP (Fig.
26-32a); the phosphate groups add negative charges to these polypeptides. The functional significance of thismodification varies from one protein to the next. For example, the milkprotein casein has many phosphoserine groups, which function to bindCa 2+ . Given that Ca 2+ and phosphate, as well as amino acids, arerequired by suckling young, casein provides three essential nutrients.The phosphorylation and dephosphorylation of the hydroxyl group ofcertain Ser residues is required to regulate the activity of some enzymes, such as glycogen phosphorylase (see Fig.
14-17). Phosphorylation of specific Tyr residues of some proteins is an important step in thetransformation of normal cells into cancer cells (see Fig. 22-37).Extra carboxyl groups may be added to Asp and Glu residues ofsome proteins. For example, the blood-clotting protein prothrombincontains a number of y-carboxyglutamate residues (Fig. 26-32b) in itsamino-terminal region, introduced by a vitamin K-requiring enzyme.These groups bind Ca 2+ , required to initiate the clotting mechanism.In some proteins certain Lys residues are methylated enzymatically (Fig. 26-32c).
Monomethyl- and dimethyllysine residues arepresent in some muscle proteins and in cytochrome c. The calmodulinof most organisms contains one trimethyllysine residue at a specificposition. In other proteins the carboxyl groups of some Glu residuesundergo methylation (Fig. 26-32c), which removes their negativecharge.Attachment of Carbohydrate Side Chains The carbohydrate sidechains of glycoproteins are attached covalently during or after the synthesis of the polypeptide chain. In some glycoproteins the carbohydrateside chain is attached enzymatically to Asn residues (AMinked oligosaccharides), in others to Ser or Thr residues (O-linked oligosaccharides; see Fig. 11-23).
Many proteins that function extracellularly, aswell as the "lubricating" proteoglycans coating mucous membranes,contain oligosaccharide side chains (see Fig. 11-21).Addition of Isoprenyl Groups A number of eukaryotic proteins areisoprenylated; a thioether bond is formed between the isoprenyl groupand a Cys residue of the protein (see Fig. 10-3). The isoprenyl groupsare derived from pyrophosphate intermediates of the cholesterol biosynthetic pathway (see Fig. 20-34), such as farnesyl pyrophosphate(Fig. 26-33). Proteins modified in this way include the products of theras oncogenes and proto-oncogenes (Chapter 22), G proteins (Chapter22), and proteins called lamins, found in the nuclear matrix.
In someChapter 26 Protein Metabolism92'Figure 26-33 Farnesylation of a Cys residue on aprotein. The thioether linkage is shown in red. Theras protein is the product of the ras oncogene.Ras proteinFarnesyl pyrophosphatePPiRas V S - C H 2cases the isoprenyl group serves to help anchor the protein in a membrane.
The transforming (carcinogenic) activity of the ras oncogene islost when isoprenylation is blocked, stimulating great interest in identifying inhibitors of this posttranslational modification pathway foruse in cancer chemotherapy.Addition of Prosthetic Groups Many prokaryotic and eukaryotic proteins require for their activity covalently bound prosthetic groups;these are attached to the polypeptide chain after it leaves the ribosome. Two examples are the covalently bound biotin molecule in acetylCoA carboxylase and the heme group of cytochrome c.Proteolytic Processing Many proteins—for example, insulin (seeFig. 22-20), some viral proteins, and proteases such as trypsin andchymotrypsin (see Fig. 8-30)—are initially synthesized as larger, inactive precursor proteins.
These precursors are proteolyticallytrimmed to produce their final, active forms.Formation of Bisulfide Cross-Links Proteins to be exported fromeukaryotic cells, after undergoing spontaneous folding into their nativeconformations, are often covalently cross-linked by the formation ofintrachain or interchain disulfide bridges between Cys residues. Thecross-links formed in this way help to protect the native conformationof the protein molecule from denaturation in an extracellular environment that can differ greatly from that inside the cell.Protein Synthesis Is Inhibited byMany Antibiotics and ToxinsProtein synthesis is a central function in cellular physiology, and assuch it is the primary target of a wide variety of naturally occurringantibiotics and toxins.
Except as noted, these antibiotics inhibit protein synthesis in bacteria. The differences between bacterial and eukaryotic protein synthesis are sufficient that most of these compoundsare relatively harmless to eukaryotic cells. Antibiotics are important"biochemical weapons," synthesized by some microorganisms and extremely toxic to others.
Antibiotics have become valuable tools in thestudy of protein synthesis; nearly every step in protein synthesis canbe specifically inhibited by one antibiotic or another.One of the best-understood inhibitory antibiotics is puromycin,made by the mold Streptomyces alboniger. Puromycin has a structureA sitepuromycinPsitepeptidyl-tRNAFigure 26-34 Puromycin resembles the aminoacylend of a charged tRNA and can bind to the ribosomal A site, where it can participate in peptide bondformation (a). The product of this reaction, insteadof being translocated to the P site, dissociates fromthe ribosome, causing premature chain termination.(b) Peptidyl puromycin.CH2-/~VoCH3VOCH 3OmRNA1 U 11 11. 11 .1.
Mvery similar to the 3' end of an aminoacyl-tRNA (Fig. 26-34). It bindsto the A site and participates in all elongation steps up to and including peptide bond formation, producing a peptidyl puromycin. However,puromycin will not bind to the P site, nor does it engage in translocation. It dissociates from the ribosome shortly after it is linked to thecarboxyl terminus of the peptide, prematurely terminating synthesisof the polypeptide.Tetracyclines inhibit protein synthesis in bacteria by blockingthe A site on the ribosome, inhibiting binding of aminoacyl-tRNAs.Chloramphenicol inhibits protein synthesis by bacterial (and mitochondrial and chloroplast) ribosomes by blocking peptidyl transfer butdoes not affect cytosolic protein synthesis in eukaryotes.
Conversely,cycloheximide blocks the peptidyl transferase of 80S eukaryotic ribosomes but not that of 70S bacterial (and mitochondrial and chloroplast) ribosomes. Streptomycin, a basic trisaccharide, causes misreading of the genetic code in bacteria at relatively low concentrationsand inhibits initiation at higher concentrations.Several other inhibitors of protein synthesis are notable because oftheir toxicity to humans and other mammals. Diphtheria toxin(Mr 65,000) catalyzes the ADP-ribosylation of a diphthamide (a modified histidine) residue on eukaryotic elongation factor eEF2, therebyinactivating it (see Box 8-4). Ricin, an extremely toxic protein of thecastor bean, inactivates the 60S subunit of eukaryotic ribosomes.OHNHICONH 2OHONH—C—NH2OH OTetracyclineOH-ONH—C—CHC12f~\O2NH^^=^IIOIIVCH-CH OOH CH 2OHChloramphenicol928HJOHCycloheximideCHOStreptomycinOHChapter 26 Protein Metabolism929Protein Targeting and DegradationThe eukaryotic cell is made up of many structures, compartments, andorganelles, each with specific functions requiring distinct sets of proteins and enzymes.
The synthesis of almost all these proteins begins onfree ribosomes in the cytosol. How are these proteins directed to theirfinal cellular destinations?The answer to this question is at once complex, fascinating, andunfortunately incomplete. Enough is known, however, to outline manykey steps in this process. Proteins destined for secretion, integration inthe plasma membrane, or inclusion in lysosomes generally share thefirst few steps of a transport pathway that begins in the endoplasmicreticulum. Proteins destined for mitochondria, chloroplasts, or the nucleus each use separate mechanisms, and proteins destined for thecytosol simply remain where they are synthesized.
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