Chapter 7. Control of Gene Expression
Negative and positive control of alternative RNA splicing.
(A) In negative control, a repressor protein binds to a specific sequence in the premRNA transcript and blocks access of the splicing machinery to a splice junction. This often results in the use of a secondary splice site, thereby producing an altered pattern of splicing (see Figure 7-56). (B) In positive control, the splicing machinery is unable to remove a particular intron sequence efficiently without assistance from an activator protein. Because RNA is flexible, the nucleotide sequences that bind these activators can be located many nucleotide pairs from the splice junctions they control, and they are often called splicing enhancers, by analogy with the transcriptional enhancers mentioned earlier in this chapter.
Regulation of Gene Expression by Noncoding RNAs Highlights
*Small Noncoding RNA Transcripts Regulate Many Animal and Plant Genes Through RNA Interference *miRNAs Regulate mRNA Translation and Stability *RNA Interference Is Also Used as a Cell Defense Mechanism *RNA Interference Can Direct Heterochromatin Formation *RNA Interference Has Become a Powerful Experimental Tool *Bacteria Use Small Noncoding RNAs to Protect Themselves from Viruses *Long Noncoding RNAs Have Diverse Functions in the Cell
An Overview of Gene Control Highlights
*The Different Cell Types of a Multicellular Organism Contain the Same DNA *Different Cell Types Synthesize Different Sets of RNAs and Proteins *External Signals Can Cause a Cell to Change the Expression of Its Genes *Gene Expression Can Be Regulated at Many of the Steps in the Pathway from DNA to RNA to Protein * * *
Control Of Transcription By Sequence Specific DNA-Binding Proteins Highlights
*The Sequence of Nucleotides in the DNA Double Helix Can Be Read by Proteins *Transcription Regulators Contain Structural Motifs That Can Read DNA Sequences *Dimerization of Transcription Regulators Increases Their Affinity and Specificity for DNA *Transcription Regulators Bind Cooperatively to DNA *Nucleosome Structure Promotes Cooperative Binding of Transcription Regulators
Transcription Regulators Switch Genes On And Off Highlights
*The Tryptophan Repressor Switches Genes Off *Repressors Turn Genes Off and Activators Turn Them On *An Activator and a Repressor Control the Lac Operon *DNA Looping Can Occur During Bacterial Gene Regulation *Complex Switches Control Gene Transcription in Eukaryotes *A Eukaryotic Gene Control Region Consists of a Promoter Plus Many cis-Regulatory Sequences *Eukaryotic Transcription Regulators Work in Groups *Activator Proteins Promote the Assembly of RNA Polymerase at the Start Point of Transcription *Eukaryotic Transcription Activators Direct the Modification of Local Chromatin Structure *Transcription Activators Can Promote Transcription by Releasing RNA Polymerase from Promoters *Transcription Activators Work Synergistically *Insulator DNA Sequences Prevent Eukaryotic Transcription Regulators from Influencing Distant Genes
Post-Transcriptional Controls Highlights
*Transcription Attenuation Causes the Premature Termination of Some RNA Molecules *Alternative RNA Splicing Can Produce Different Forms of a Protein from the Same Gene *The Definition of a Gene Has Been Modified Since the Discovery of Alternative RNA Splicing * A Change in the Site of RNA Transcript Cleavage and Poly-A Addition Can Change the C-terminus of a Protein *RNA Editing Can Change the Meaning of the RNA Message *RNA Transport from the Nucleus Can Be Regulated *Some mRNAs Are Localized to Specific Regions of the Cytosol *The 5ʹ and 3ʹ Untranslated Regions of mRNAs Control Their Translation *The Phosphorylation of an Initiation Factor Regulates Protein Synthesis Globally *Initiation at AUG Codons Upstream of the Translation Start Can Regulate Eukaryotic Translation Initiation *Changes in mRNA Stability Can Regulate Gene Expression
Gene expression in eukaryotes
- Transcription occurs in the nucleus - RNA is processed - RNA is moved to the cytoplasm - Translation occurs in the cytoplasm
MOLECULAR GENETIC MECHANISMS THAT CREATE AND MAINTAIN SPECIALIZED CELL TYPES
Although all cells must be able to switch genes on and off in response to changes in their environments, the cells of multicellular organisms have evolved this capacity to an extreme degree. In particular, once a cell in a multicellular organism becomes committed to differentiate into a specific cell type, the cell maintains this choice through many subsequent cell generations, which means that it remembers the changes in gene expression involved in the choice. This phenomenon of cell memory is a prerequisite for the creation of organized tissues and for the maintenance of stably differentiated cell types. In contrast, other changes in gene expression in eukaryotes, as well as most such changes in bacteria, are only transient. The tryptophan repressor, for example, switches off the tryptophan genes in bacteria only in the presence of tryptophan; as soon as tryptophan is removed from the medium, the genes are switched back on, and the descendants of the cell will have no memory that their ancestors had been exposed to tryptophan. In this section, we shall examine not only cell memory mechanisms, but also how gene regulatory devices can be combined to create the "logic circuits" through which cells integrate signals and remember events in their past. We begin by considering one such complex gene control region in detail.
RNA Interference Has Become a Powerful Experimental Tool
Although it likely arose as a defense mechanism against viruses and transposable elements, RNA interference, as we have seen, has become thoroughly integrated into many aspects of normal cell biology, ranging from the control of gene expression to the structure of chromosomes. It has also been developed by scientists into a powerful experimental tool that allows almost any gene to be inactivated by evoking an RNAi response to it. This technique, which can be readily carried out in cultured cells and, in many cases, whole animals and plants, has made possible new genetic approaches in cell and molecular biology. We shall discuss it in detail in the following chapter where we cover modern genetic methods used to study cells (see pp. 499-501). RNAi also has potential in treating human disease. Since many human disorders result from the misexpression of genes, the ability to turn these genes off by experimentally introducing complementary siRNA molecules holds great medical promise. Although the mechanism of RNA interference was discovered a few decades ago, we are still being surprised by its mechanistic details and by its broad biological implications.
Genes can be switched on by activator proteins.
An activator protein binds to its cis-regulatory sequence on the DNA and interacts with the RNA polymerase to help it initiate transcription. Without the activator, the promoter fails to initiate transcription efficiently. In bacteria, the binding of the activator to DNA is often controlled by the interaction of a metabolite or other small molecule (red triangle) with the activator protein. The Lac operon works in this manner, as we discuss shortly.
TRANSCRIPTION REGULATORS SWITCH GENES ON AND OFF
Having seen how transcription regulators bind to cis-regulatory sequences embedded in the genome, we can now discuss how, once bound, these proteins influence the transcription of genes. The situation in bacteria is simpler than in eukaryotes (for one thing, chromatin structure is not an issue), and we therefore discuss it first. Following this, we turn to the more complex situation in eukaryotes.
CONTROL OF TRANSCRIPTION BY SEQUENCE SPECIFIC DNA-BINDING PROTEINS
How does a cell determine which of its thousands of genes to transcribe? Perhaps the most important concept, one that applies to all species on Earth, is based on a group of proteins known as transcription regulators. These proteins recognize specific sequences of DNA (typically 5-10 nucleotide pairs in length) that are often called cis-regulatory sequences, because they must be on the same chromosome (that is, in cis) to the genes they control.
Genes can be switched off by repressor proteins.
If the concentration of tryptophan inside a bacterium is low (left), RNA polymerase (blue) binds to the promoter and transcribes the five genes of the tryptophan operon. However, if the concentration of tryptophan is high (right), the repressor protein (dark green) becomes active and binds to the operator (light green), where it blocks the binding of RNA polymerase to the promoter. Whenever the concentration of intracellular tryptophan drops, the repressor falls off the DNA, allowing the polymerase to again transcribe the operon. Although not shown in the figure, the repressor is a stable dimer.
POST-TRANSCRIPTIONAL CONTROLS
In principle, every step required for the process of gene expression can be controlled. Indeed, one can find examples of each type of regulation, and many genes are regulated by multiple mechanisms. As we have seen, controls on the initiation of gene transcription are a critical form of regulation for all genes. But other controls can act later in the pathway from DNA to protein to modulate the amount of gene product that is made—and in some cases, to determine the exact amino acid sequence of the protein product. These post-transcriptional controls, which operate after RNA polymerase has bound to the gene's promoter and has begun RNA synthesis, are crucial for the regulation of many genes. In the following sections, we consider the varieties of post-transcriptional regulation in temporal order, according to the sequence of events that an RNA molecule might experience after its transcription has begun
REGULATION OF GENE EXPRESSION BY NONCODING RNAs
In the previous chapter, we introduced the central dogma, according to which the flow of genetic information proceeds from DNA through RNA to protein (Figure 6-1). But we have seen throughout this book that RNA molecules perform many critical tasks in the cell besides serving as intermediate carriers of genetic information. Among these noncoding RNAs are the rRNA and tRNA molecules, which are responsible for reading the genetic code and synthesizing proteins. The RNA molecule in telomerase serves as a template for the replication of chromosome ends, snoRNAs modify ribosomal RNA, and snRNAs carry out the major events of RNA splicing. And we saw in the previous section that Xist RNA has an important role in inactivating one copy of the X chromosome in females. A series of recent discoveries has revealed that noncoding RNAs are even more prevalent than previously imagined. We now know that such RNAs play widespread roles in regulating gene expression and in protecting the genome from viruses and transposable elements. These newly discovered RNAs are the subject of this section.
Transcription attenuation
Inhibition of gene expression by the premature termination of transcription. The expression of some genes is inhibited by premature termination of transcription, a phenomenon called transcription attenuation.
Post-Transcriptional Controls Summary
Many steps in the pathway from RNA to protein are regulated by cells in order to control gene expression. Most genes are regulated at multiple levels, in addition to being controlled at the initiation stage of transcription. The regulatory mechanisms include (1) attenuation of the RNA transcript by its premature termination, (2) alternative RNA splice-site selection, (3) control of 3ʹ-end formation by cleavage and poly-A addition, (4) RNA editing, (5) control of transport from the nucleus to the cytosol, (6) localization of mRNAs to particular parts of the cell, (7) control of translation initiation, and (8) regulated mRNA degradation. Most of these control processes require the recognition of specific sequences or structures in the RNA molecule being regulated, a task performed by either regulatory proteins or regulatory RNA molecules.
RNA editing
Process that alters the nucleotide sequences of RNA transcripts once they are synthesized and thereby changes the coded message they carry.
Regulation of Gene Expression by Noncoding RNAs Summary
RNA molecules have many uses in the cell besides carrying the information needed to specify the order of amino acids during protein synthesis. Although we have encountered noncoding RNAs in other chapters (tRNAs, rRNAs, snoRNAs, for example), the sheer number of noncoding RNAs produced by cells has surprised scientists. One well understood use of noncoding RNAs occurs in RNA interference, where guide RNAs (miRNAs, siRNAs, piRNAs) base-pair with mRNAs. RNA interference can cause mRNAs to be either destroyed or translationally repressed. It can also cause specific genes to be packaged into heterochromatin suppressing their transcription. In bacteria and archaebacteria, RNA interference is used as an adaptive immune response to destroy viruses that infect them. A large family of large noncoding RNAs (lncRNAs) has recently been discovered. Although the function of most of these RNAs is unknown, some serve as RNA scaffolds to bring specific proteins and RNA molecules together to speed up needed reactions.
RNA interference in eukaryotes.
Single-stranded interfering RNAs are generated from double-stranded RNA. They locate target RNAs through base-pairing and, at this point, several fates are possible, as shown. As described in the text, there are several types of RNA interference; the way the double-stranded RNA is produced and processed and the ultimate fate of the target RNA depends on the particular system.
AN OVERVIEW OF GENE CONTROL
The different cell types in a multicellular organism differ dramatically in both structure and function. If we compare a mammalian neuron with a liver cell, for example, the differences are so extreme that it is difficult to imagine that the two cells contain the same genome (Figure 7-1). For this reason, and because cell differentiation often seemed irreversible, biologists originally suspected that genes might be selectively lost when a cell differentiates. We now know, however, that cell differentiation generally occurs without changes in the nucleotide sequence of a cell's genome.
Post-transcriptional controls of gene expression.
The final synthesis rate of a protein can, in principle, be controlled at any of the steps listed in capital letters. In addition, RNA splicing, RNA editing, and translation recoding can also alter the sequence of amino acids in a protein, making it possible for the cell to produce more than one protein variant from the same gene. Only a few of the steps depicted here are likely to be critical for the regulation of any one particular protein.
An Overview of Gene Control Summary
The genome of a cell contains in its DNA sequence the information to make many thousands of different protein and RNA molecules. A cell typically expresses only a fraction of its genes, and the different types of cells in multicellular organisms arise because different sets of genes are expressed. Moreover, cells can change the pattern of genes they express in response to changes in their environment, such as signals from other cells. Although all of the steps involved in expressing a gene can in principle be regulated, for most genes the initiation of RNA transcription provides the most important point of control.
Molecular Genetic Mechanisms That Create And Maintain Specialized Cell Types Summary
The many types of cells in animals and plants are created largely through mechanisms that cause different sets of genes to be transcribed in different cells. The transcription of any particular gene is generally controlled by a combination of transcription regulators. Each type of cell in a higher eukaryotic organism contains a specific set of transcription regulators that ensures the expression of only those genes appropriate to that type of cell. A given transcription regulator may be active in a variety of circumstances and is typically involved in the regulation of many different genes. Since specialized animal cells can maintain their unique character through many cell-division cycles, and even when grown in culture, the gene regulatory mechanisms involved in creating them must be stable once established and heritable when the cell divides. These features reflect the cell's memory of its developmental history. Direct or indirect positive feedback loops, which enable transcription regulators to perpetuate their own synthesis, provide the simplest mechanism for cell memory. Transcription circuits also provide the cell with the means to carry out other types of logic operations. Simple transcription circuits combined into large regulatory networks drive highly sophisticated programs of embryonic development that will require new approaches to decipher.
miRNA processing and mechanism of action.
The precursor miRNA, through complementarity between one part of its sequence and another, forms a double-stranded structure. This RNA is cropped while still in the nucleus and then exported to the cytosol, where it is further cleaved by the Dicer enzyme to form the miRNA proper. Argonaute, in conjunction with other components of RISC, initially associates with both strands of the miRNA and then cleaves and discards one of them. The other strand guides RISC to specific mRNAs through base-pairing. If the RNA-RNA match is extensive, as is commonly seen in plants, Argonaute cleaves the target mRNA, causing its rapid degradation. In mammals, the miRNA-mRNA match often does not extend beyond a short seven-nucleotide "seed" region near the 5ʹ end of the miRNA. This less extensive base-pairing leads to inhibition of translation, mRNA destabilization, and transfer of the mRNA to P-bodies, where it is eventually degraded.
How cells control the proteins it makes
There are many steps in the pathway leading from DNA to protein. We now know that all of them can in principle be regulated. Thus a cell can control the proteins it makes by: 1. Transcriptional control: controlling when and how often a given gene is transcribed 2. RNA processing control: controlling the splicing and processing of RNA transcripts 3. RNA transport and localization control: selecting which completed mRNAs are exported from the nucleus to the cytosol and determining where in the cytosol they are localized 4. Translational control: selecting which mRNAs in the cytoplasm are translated by ribosomes 5. mRNA degradation control: selectively destabilizing certain mRNA molecules in the cytoplasm 6. Protein activity control: selectively activating, inactivating, degrading, or localizing specific protein molecules after they have been made
Control Of Transcription By Sequence Specific DNA-Binding Proteins Summary
Transcription regulators recognize short stretches of double-helical DNA of defined sequence called cis-regulatory sequences, and thereby determine which of the thousands of genes in a cell will be transcribed. Approximately 10% of the protein-coding genes in most organisms produce transcription regulators, and they control many features of cells. Although each of these transcription regulators has unique features, most bind to DNA as homodimers or heterodimers and recognize DNA through one of a small number of structural motifs. Transcription regulators typically work in groups and bind DNA cooperatively, a feature that has several underlying mechanisms, some of which exploit the packaging of DNA in nucleosomes.
Transcription Regulators Switch Genes On And Off Summary
Transcription regulators switch the transcription of individual genes on and off in cells. In prokaryotes, these proteins typically bind to specific DNA sequences close to the RNA polymerase start site and, depending on the nature of the regulatory protein and the precise location of its binding site relative to the start site, either activate or repress transcription of the gene. The flexibility of the DNA helix, however, also allows proteins bound at distant sites to affect the RNA polymerase at the promoter by the looping out of the intervening DNA. The regulation of higher eukaryotic genes is much more complex, commensurate with a larger genome size and the large variety of cell types that are formed. A single eukaryotic gene is typically controlled by many transcription regulators bound to sequences that can be tens or even hundreds of thousands of nucleotide pairs from the promoter that directs transcription of the gene. Eukaryotic activators and repressors act by a wide variety of mechanisms—generally altering chromatin structure and controlling the assembly of the general transcription factors and RNA polymerase at the promoter. They do this by attracting coactivators and co-repressors, protein complexes that perform the necessary biochemical reactions. The time and place that each gene is transcribed, as well as its rates of transcription under different conditions, are determined by the particular spectrum of transcription regulators that bind to the regulatory region of the gene.
Gene expression
is the process by which the instructions in our DNA are converted into a functional product, such as a protein.