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Gene Expression Can Be Controlled at Posttranscriptional Levels

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Gene Expression Can Be Controlled at Posttranscriptional Levels
Figure 31.32. Domain Structure of CREB-Binding Protein (CBP). The CREB-binding protein includes at least three
types of protein-protein interaction domains in addition to a histone acetyltransferase domain that lies near the
carboxyl terminus. The kinase-inducible interaction (KIX) domain interacts specifically with a region of CREB in
its phosphorylated form.
III. Synthesizing the Molecules of Life
31. The Control of Gene Expression
31.3. Transcriptional Activation and Repression Are Mediated by Protein-Protein Interactions
Figure 31.33. Interaction between CBP and CREB. The KIX domain of CBP binds a region of CREB in its
phosphorylated form.
III. Synthesizing the Molecules of Life
31. The Control of Gene Expression
31.4. Gene 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.
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.
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.
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).
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.
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.
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.
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 ironsensing 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.
III. Synthesizing the Molecules of Life
31. The Control of Gene Expression
31.4. Gene Expression Can Be Controlled at Posttranscriptional Levels
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 region (blue and red nucleotides). (B and C) The attenuator region can adopt two distinct
stem-loop structures.
III. Synthesizing the Molecules of Life
31. The Control of Gene Expression
31.4. Gene Expression Can Be Controlled at Posttranscriptional Levels
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 Trp-tRNA, giving time for an alternative RNA structure to form that does not
terminate transcription efficiently.
III. Synthesizing the Molecules of Life
31. The Control of Gene Expression
31.4. Gene Expression Can Be Controlled at Posttranscriptional Levels
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 leads to attenuation.
III. Synthesizing the Molecules of Life
31. The Control of Gene Expression
31.4. Gene Expression Can Be Controlled at Posttranscriptional Levels
Figure 31.37. Structure of Ferritin. (A) Twenty-four ferritin polypeptides form a nearly spherical shell. (B) A cutaway view reveals the core that stores iron as an iron oxide-hydroxide complex.
III. Synthesizing the Molecules of Life
31. The Control of Gene Expression
31.4. Gene Expression Can Be Controlled at Posttranscriptional Levels
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
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