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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 31.4Gene Expression Can Be Controlled at Posttranscriptional Levels

Modulation of the rate of transcriptional initiation is the most common mechanism of gene regulation. However, other stages of transcription also are targets for regulation in some cases. In addition, the process of translation provides other points of intervention for regulating the level of a protein produced in a cell. These mechanisms are quite distinct in prokaryotic and eukaryotic cells because prokaryotes and eukaryotes differ greatly in how transcription and translation are coupled and in how translation is initiated. We shall consider two important examples of posttranscriptional regulation: one from prokaryotes and the other from eukaryotes. In both examples, regulation depends on the formation of distinct secondary structures in mRNA.

31.4.1 Attenuation Is a Prokaryotic Mechanism for Regulating Transcription Through Modulation of Nascent RNA Secondary Structure

A novel mechanism for regulating transcription in bacteria was discovered by Charles Yanofsky and his colleagues as a result of their studies of the tryptophan operon. The 7-kb mRNA transcript from this operon encodes five enzymes that convert chorismate into tryptophan (Section 24.2.10). The mode of regulation of this operon is called attenuation, and it depends on features at the 5′ end of the mRNA product (Figure 31.34). Upstream of the coding regions for the enzymes responsible for tryptophan biosynthesis lies a short open reading frame encoding a 14-amino-acid leader peptide. Following this open reading frame is a region of RNA, called an attenuator, that is capable of forming several alternative structures. Recall that transcription and translation are tightly coupled in bacteria. Thus, the translation of the trp mRNA begins soon after the ribosome-binding site has been synthesized.

Figure 31.34. Leader Region of TRP mRNA.

Figure 31.34

Leader Region of TRP mRNA. (A) The nucleotide sequence of the 5′ end of trp mRNA includes a short open reading frame that encodes a peptide comprising 14 amino acids; the leader encodes two tryptophan residues and has an untranslated attenuator (more...)

A ribosome is able to translate the leader region of the mRNA product only in the presence of adequate concentrations of tryptophan. When enough tryptophan is present, a stem-loop structure forms in the attenuator region, which leads to the release of RNA polymerase from the DNA (Figure 31.35). However, when tryptophan is scarce, transcription is terminated less frequently. How does the level of tryptophan alter transcription of the trp operon? An important clue was the finding that the 14-amino-acid leader peptide includes two adjacent tryptophan residues. When tryptophan is scarce, little tryptophanyl-tRNA is present. Thus, the ribosome stalls at the tandem UGG codons encoding tryptophan. This delay leaves the adjacent region of the mRNA exposed as transcription continues. An alternative RNA structure that does not function as a terminator is formed and transcription continues into and through the coding regions for the enzymes. Thus, attenuation provides an elegant means of sensing the supply of tryptophan required for protein synthesis.

Figure 31.35. Attenuation.

Figure 31.35

Attenuation. (A) In the presence of adequate concentrations of tryptophan (and, hence, Trp-tRNA), translation proceeds rapidly and an RNA structure forms that terminates transcription. (B) At low concentrations of tryptophan, translation stalls awaiting (more...)

Several other operons for the biosynthesis of amino acids in E. coli also are regulated by attenuator sites. The leader peptide of each contains an abundance of the amino acid residues of the type controlled by the operon (Figure 31.36). For example, the leader peptide for the phenylalanine operon includes 7 phenylalanine residues among 15 residues. The threonine operon encodes enzymes required for the synthesis of both threonine and isoleucine; the leader peptide contains 8 threonine and 4 isoleucine residues in a 16-residue sequence. The leader peptide for the histidine operon includes 7 histidine residues in a row. In each case, low levels of the corresponding charged tRNA causes the ribosome to stall, trapping the nascent mRNA in a state that can form a structure that allows RNA polymerase to read through the attenuator site.

Figure 31.36. Leader Peptide Sequences.

Figure 31.36

Leader Peptide Sequences. Amino acid sequences and the corresponding mRNA nucleotide sequences of the (A) threonine operon, (B) phenylalanine operon, and (C) histidine operon. In each case, an abundance of one amino acid in the leader peptide sequence (more...)

31.4.2 Genes Associated with Iron Metabolism Are Translationally Regulated in Animals

RNA secondary structure plays a role in the regulation of iron metabolism in eukaryotes. Iron is an essential nutrient, required for the synthesis of hemoglobin, cytochromes, and many other proteins. However, excess iron can be quite harmful because, untamed by a suitable protein environment, iron can initiate a range of free-radical reactions that damage proteins, lipids, and nucleic acids. Animals have evolved sophisticated systems for the accumulation of iron in times of scarcity and for the safe storage of excess iron for later use. Key proteins include transferrin, a transport protein that carries iron in the serum, transferrin receptor, a membrane protein that binds iron-loaded transferrin and initiates its entry into cells, and ferritin, an impressively efficient iron-storage protein found primarily in the liver and kidneys. Twenty-four ferritin polypeptides form a nearly spherical shell that encloses as many as 2400 iron atoms, a ratio of one iron atom per amino acid (Figure 31.37).

Figure 31.37. Structure of Ferritin.

Figure 31.37

Structure of Ferritin. Image mouse.jpg (A) Twenty-four ferritin polypeptides form a nearly spherical shell. (B) A cut-away view reveals the core that stores iron as an iron oxide-hydroxide complex.

Ferritin and transferrin receptor expression levels are reciprocally related in their responses to changes in iron levels. When iron is scarce, the amount of transferrin receptor increases and little or no new ferritin is synthesized. Interestingly, the extent of mRNA synthesis for these proteins does not change correspondingly. Instead, regulation takes place at the level of translation.

Consider ferritin first. Ferritin mRNA includes a stem-loop structure termed an iron-response element (IRE) in its 5′ untranslated region (Figure 31.38). This stem-loop binds a 90-kd protein, called an IRE-binding protein (IRE-BP), that blocks the initiation of translation. When the iron level increases, the IRE-BP binds iron as a 4Fe-4S cluster. The IRE-BP bound to iron cannot bind RNA, because the binding sites for iron and RNA substantially overlap. Thus, in the presence of iron, ferritin mRNA is released from the IRE-BP and translated to produce ferritin, which sequesters the excess iron.

Figure 31.38. Iron-Response Element.

Figure 31.38

Iron-Response Element. Ferritin mRNA includes a stem-loop structure, termed an iron-response element (IRE), in its 5′ untranslated region. The IRE binds a specific protein that blocks the translation of this mRNA under low iron conditions.

An examination of the nucleotide sequence of transferrin-receptor mRNA reveals the presence of several IRE-like regions. However, these regions are located in the 3′ untranslated region rather than in the 5′ untranslated region (Figure 31.39). Under low-iron conditions, IRE-BP binds to these IREs. However, given the location of these binding sites, the transferrin-receptor mRNA can still be translated. What happens when the iron level increases and the IRE-BP no longer binds transferrin-receptor mRNA? Freed from the IRE-BP, transferrin-receptor mRNA is rapidly degraded. Thus, an increase in the cellular iron level leads to the destruction of transferrin-receptor mRNA and, hence, a reduction in the production of transferrin-receptor protein.

Figure 31.39. Transferrin-receptor mRNA.

Figure 31.39

Transferrin-receptor mRNA. This mRNA has a set of ironresponse elements (IREs) in its 3′ untranslated region. The binding of the IRE-binding protein to these elements stabilizes the mRNA but does not interfere with translation.

Image tree.jpg The purification of the IRE-BP and the cloning of its cDNA were sources of truly remarkable insight into evolution. The IRE-BP was found to be approximately 30% identical in amino acid sequence with the citric acid cycle enzyme aconitase from mitochondria. Further analysis revealed that the IRE-BP is, in fact, an active aconitase enzyme; it is a cytosolic aconitase that had been known for a long time, but its function was not well understood (Figure 31.40). The iron-sulfur center at the active site of the IRE-BP is rather unstable, and loss of the iron triggers significant changes in protein conformation. Thus, this protein can serve as an iron-sensing factor.

Figure 31.40. The IRE-BP Is an Aconitase.

Figure 31.40

The IRE-BP Is an Aconitase. Image mouse.jpg (A) Aconitase contains a relatively unstable 4Fe-4S cluster at its center. (B) Under conditions of low iron, the 4Fe-4S cluster dissociates and appropriate RNA molecules can bind in its place.

Other mRNAs, including those taking part in heme synthesis, have been found to contain IREs. Thus, genes encoding proteins required for iron metabolism acquired sequences that, when transcribed, provided binding sites for the iron-sensing protein. An environmental signal—the concentration of iron—controls the translation of proteins required for the metabolism of this metal. The IREs have evolved appropriately in the untranslated regions of mRNAs to lead to beneficial regulation by iron levels.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22400


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