Many proteins are synthesized on polysomes in the cytosol. After they are released from ribosomes, they remain in the cytosol where they carry out their functions.
Other proteins synthesized on cytosolic ribosomes enter organelles, such as mi- tochondria or nuclei. These proteins contain amino acid sequences called target- ing sequences or signal sequences that facilitate their transport into a certain organelle. Another group of proteins are synthesized on ribosomes bound to the rough endoplasmic reticulum (RER). These proteins are destined for secretion or for incorporation into various subcellular organelles (e.g., lysosomes, endoplas- mic reticulum [ER], Golgi complex) or cellular membranes, including the plasma membrane.
Proteins that enter the RER as they are being synthesized have signal peptides near their N-termini, which do not have a common amino acid sequence. How- ever, they do contain several hydrophobic residues and are 15 to 30 amino acids in length (Fig. 12.9). A signal recognition particle (SRP) binds to the ribosome and to the signal peptide as the nascent polypeptide emerges from the tunnel in the ribo- some and translation ceases. When the SRP subsequently binds to an SRP receptor (docking protein) on the RER, translation resumes, and the polypeptide begins to enter the lumen of the RER. The signal peptide is removed by the signal peptidase and the remainder of the newly synthesized protein enters the lumen of the RER.
These proteins are transferred in small vesicles to the Golgi complex.
The Golgi complex serves to process the proteins it receives from the RER and to sort them so that they are delivered to their appropriate destinations.
Processing, which can be initiated in the ER, involves glycosylation, the addition of carbohydrate groups, and modifi cation of existing carbohydrate chains. Sorting signals permit delivery of proteins to their target locations. For example, glycosyl- ation of enzymes destined to become lysosomal enzymes results in the presence of a mannose 6- phosphate residue on an oligosaccharide attached to the enzyme. This residue is recognized by the mannose 6-phosphate receptor protein, which incor- porates the enzyme into a clathrin-coated vesicle. The vesicle travels to endosomes and is eventually incorporated into lysosomes. Other proteins containing a KDEL (lys-asp-glu-leu) sequence at their carboxyl terminal are returned to the ER from the Golgi. Proteins with hydrophobic regions can embed in various membranes.
Some proteins, whose sorting signals have not yet been determined, enter secretory vesicles and travel to the cell membrane where they are secreted by the process of exocytosis.
Table 12.3 Posttranslational Modifi cations of Proteins Acetylation
ADP-ribosylation Carboxylation Fatty acylation Glycosylation Hydroxylation Methylation Phosphorylation Prenylation
I-cell disease (mucolipidosis II) is an inherited recessive disorder of protein targeting. Lysosomal pro- teins are not sorted properly from the Golgi to the lysosomes, and lysosomal enzymes end up secreted from the cell. This is due to a muta- tion in the enzyme N-acetylglucosamine phos- photransferase, which is a required fi rst step for attaching the lysosomal targeting signal, mannose 6-phosphate, to lysosomal proteins.
Thus, lysosomal proteins cannot be targeted to the lysosomes, and these organelles be- come clogged with materials that cannot be digested, destroying overall lysosomal func- tion. This leads to a lysosomal storage disease of severe consequence, with death before the age of 8 years.
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CHAPTER 12 ■ TRANSLATION: SYNTHESIS OF PROTEINS 187
1
2 3
4 5 6
3' 5'
mRNA 60S 40S
60S 40S
Signal peptide Signal-recognition
particle (SRP)
SRP
receptor Signal
peptidase Cleaved signal
peptide
Nascent protein
Completed protein Pore
RER membrane
FIG. 12.9. Synthesis of proteins on the RER. (1) Translation of the protein begins in the cytosol. (2) As the signal peptide emerges from the ri- bosome, an SRP binds to it and to the ribosome and inhibits further synthesis of the protein. (3) The SRP binds to the SRP receptor in the RER membrane, docking the ribosome on the RER. (4) The SRP is released and protein synthesis resumes. (5) As the signal peptide moves through a pore into the RER, a signal peptidase removes the signal peptide. (6) Synthesis of the nascent protein continues, and the completed protein is released into the lumen of the RER.
Table 12.4 Diseases Discussed in Chapter 12 Disorder or
Condition
Genetic or
Environmental Comments
β-Thalassemia Genetic Lisa N. has β-thalassemia intermedia, indicating that the β-globin gene product is produced at reduced levels as compared to the α-globin gene product.
This can happen by a variety of mutations.
Tay-Sachs disease
Genetic Mutation in a gene encoding a lysosomal enzyme, leading to loss of lysosomal function and death at an early age for the patient.
Diphtheria Environmental Diphtheria toxin catalyzes the ADP-ribosylation of eEF2, a necessary factor for eukaryotic protein syn- thesis. This results in cell death. Vaccination against diphtheria toxin will prevent the enzymatic actions of the toxin.
I-cell disease Genetic Mutation in posttranslational processing that leads to mistargeting of enzymes destined for the lysosomes.
Disease leads to lysosomal dysfunction and early death.
C L I N I CA L CO M M E N T S Diseases discussed in this chapter are summarized in Table 12.4.
Lisa N. Lisa N. has a β⫹-thalassemia classifi ed clinically as β-thalassemia intermedia. She produces an intermediate amount of functional β-globin chains (her hemoglobin is 7 g/dL; normal is 12 to 16 g/dL). In β0-thalasse- mia, little or none of the hemoglobin β-chain is produced. β-Thalassemia intermedia is usually the result of two different mutations (one that mildly affects the rate of syn- thesis of β-globin and one that severely affects its rate of synthesis), or, less frequently, homozygosity for a mild mutation in the rate of synthesis or a complex combination of mutations. The mutations that cause the thalassemias have been studied extensively and some of these are summarized in Table A12.1 of the online material .
Jay S. The molecular biology–genetics laboratory’s report on Jay S.’s white blood cells revealed that he had a defi ciency of hexosaminidase A caused by a defect in the gene encoding the α-subunit of this enzyme (vari-
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R E V I E W Q U E ST I O N S - C H A P T E R 12
1. In the readout of the genetic code in prokaryotes, which one of the following processes acts before any of the others?
A. tRNAi alignment with mRNA B. Termination of transcription
C. Movement of the ribosome from one codon to the next D. Recruitment of termination factors to the A site E. Export of mRNA from the nucleus
2. tRNA charged with cysteine can be chemically treated so that the amino acid changes its identity to alanine. If some of this charged tRNA is added to a protein-synthesizing extract that contains all the normal components required for translation, which of the following statements represents the most likely outcome after adding a sample of mRNA that has both Cys and Ala codons in the normal reading frame?
A. Cysteine would be added each time the alanine codon was translated.
B. Alanine would be added each time the cysteine codon was translated.
C. The protein would have a defi ciency of cysteine residues.
D. The protein would have a defi ciency of alanine residues.
E. The protein would be entirely normal.
3. The genetic code is said to be degenerate because of which one of the following?
A. All triplets seem to have at least one uracil.
B. There is wobble in the bond between the fi rst base of the anticodon and the third base of the codon.
C. Some triplets are made up of repeating purines or pyrimidines.
D. Many codons have pairs of identical bases next to each other.
E. Many of the amino acids have more than one triplet code.
4. The reason there are 64 possible codons is most likely which one of the following?
A. There are 64 aminoacyl tRNA synthetases.
B. There are four possible bases at each of three codon positions
C. Each base is able to participate in wobbling.
D. All possible reading frames can be used this way.
E. The more codons, the faster protein synthesis can be accomplished.
5. Repeating dinucleotide sequences are very common in eu- karyotic genomes (e.g., . . . ACACACACACACACACA- CACAC . . . ). Based on what you know, which one of the following statements is likely to be correct?
A. When occurring within genes, they will give rise to a monotonous run of a single amino acid.
B. Irrespective of reading frame, they will produce a run of the same two alternating amino acids.
C. Depending on the reading frame, they will give rise to repetitive runs of 1, 2, or 3 amino acids.
D. Ribosomes will rapidly dissociate from mRNAs containing such sequences.
E. If located within introns, they will initiate alternative splicing events.
ant B, Tay-Sachs disease). Hexosaminidases are lysosomal enzymes necessary for the normal degradation of glycosphingolipids such as the gangliosides. Gangliosides are found in high concentrations in neural ganglia, although they are produced in many areas of the nervous system. When the activity of these degradative enzymes is absent or subnormal, partially degraded gangliosides accumulate in lysosomes in various cells of the central nervous system, causing a wide array of neurological disorders known collectively as gangliosidoses. When the enzyme defi ciency is severe, symptoms appear within the fi rst 3 to 5 months of life. Eventually, symptoms include upper and lower motor neuron defi cits, visual diffi culties that can progress to blindness, seizures, and increasing cognitive dysfunction. By the second year of life, the patient may regress into a completely vegetative state, often succumbing to bronchopneumonia caused by aspiration and an inability to cough.
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189
13 Regulation of Gene Expression
C H A P T E R O U T L I N E
K E Y P O I N T S
■ Prokaryotic gene expression is primarily regulated at the level of initiation of gene transcription.
In general, there is one protein per gene.
■ Sets of genes encoding proteins with related functions are organized into operons.
■ Each operon is under the control of a single promoter.
■ Repressors bind to the promoter to inhibit RNA polymerase binding.
■ Activators facilitate RNA polymerase binding to the promoter.
■ Eukaryotic gene regulation occurs at several levels.
■ At the DNA structural level, chromatin must be remodeled to allow access for RNA polymerase.
■ Transcription is regulated by transcription factors, which either enhance or restrict RNA poly- merase access to the promoter.
■ RNA processing (including alternative splicing), transport from the nucleus to the cytoplasm, mRNA stability, and translation are also regulated in eukaryotes.