Now the bacterium needs to ramp up production of the lactose-digesting proteins. It does so by using an activator protein called catabolite activator protein CAP. This increases the binding ability of RNA polymerase to the promoter and ramps up transcription of the genes.
In summary, for the lac operon to be fully activated, two conditions must be met. First, the level of glucose must be very low or non-existent. Second, lactose must be present. Only when glucose is absent and lactose is present will the lac operon be transcribed maximally.
This makes sense for the cell, because it would be energetically wasteful to create the proteins to process lactose if glucose was plentiful or lactose was not available Table In eukaryotes, control of gene expression is more complex and can happen at many different levels.
Eukaryotic genes are not organized into operons, so each gene must be regulated independently. In addition, eukaryotic cells have many more genes than prokaryotic cells. For convenience, regulation is divided into five levels: epigenetic, transcriptional, post-transcriptional, translational, and post-translational Figure Epigenetics is a relatively new, but growing, field of biology.
Epigenetic control involves changes to genes that do not alter the nucleotide sequence of the DNA and are not permanent. Instead, these changes alter the chromosomal structure so that genes can be turned on or off. One example of chemical modifications of DNA is the addition of methyl groups to the DNA, in a process called methylation, In general, methylation suppresses transcription. Interestingly, methylation patterns can be passed on as cells divide.
Thus, parents may be able to pass on the tendency of a gene to be expressed in their offspring. Other heritable chemical modifications of DNA may also occur. The best-studied example of epigenetic regulation is modification of histone proteins.
Histones are chromosomal proteins that tightly wind DNA so that it fits into the nucleus of a cell. The human genome, for example, consists of over three billion nucleotide pairs. An average chromosome contains million nucleotide pairs, and each body cell contains 46 chromosomes.
If stretched out linearly, an average human chromosome would be over four centimeters long. In order to fit all of this DNA into the nucleus of a microscopic cell, the DNA must be tightly wound around proteins. It is also organized so that specific segments can be accessed as needed by a specific cell type Figure The first level of organization, or packing, is the winding of DNA strands around histone proteins.
Histones package and order DNA into structural units called nucleosome complexes, which can control the access of proteins to the DNA regions Figure Under the electron microscope, this winding of DNA around histone proteins to form nucleosomes looks like small beads on a string Figure These beads histone proteins can move along the string DNA and change the structure of the molecule.
If a gene is to be transcribed, the nucleosomes surrounding that region of DNA can slide down the DNA to open that specific chromosomal region and allow access for RNA polymerase and other proteins, called transcription factors, to bind to the promoter region and initiate transcription.
If a gene is to remain turned off, or silenced, the histone proteins and DNA have different modifications that signal a closed chromosomal configuration.
In this closed configuration, the RNA polymerase and transcription factors do not have access to the DNA and transcription cannot occur Figure How the histone proteins move is dependent on signals found on the histone proteins. These tags are not permanent, but may be added or removed as needed. Since DNA negatively charged, changes in the charge of the histone will change how tightly wound the DNA molecule will be.
When unmodified, the histone proteins have a large positive charge; by adding chemical modifications like acetyl groups, the charge becomes less positive. Transcriptional regulation is control of whether or not an mRNA is transcribed from a gene in a particular cell. Like prokaryotic cells, the transcription of genes in eukaryotes requires an RNA polymerase to bind to a promoter to initiate transcription.
In eukaryotes, RNA polymerase requires other proteins, or transcription factors , to facilitate transcription initiation. Transcription factors are proteins that bind to the promoter sequence and other regulatory sequences to control the transcription of the target gene. RNA polymerase by itself cannot initiate transcription in eukaryotic cells.
Transcription factors must bind to the promoter region first and recruit RNA polymerase to the site for transcription to begin. In eukaryotic genes, the promoter region is immediately upstream of the coding sequence. This region can range from a few to hundreds of nucleotides long. The length of the promoter is gene-specific and can differ dramatically between genes. The longer the promoter, the more available space for proteins to bind.
Consequently, the level of control of gene expression can differ quite dramatically between genes. The purpose of the promoter is to bind transcription factors that control the initiation of transcription Figure Within the promoter region, just upstream of the transcriptional start site, resides the TATA box. This box is simply a repeat of thymine and adenine dinucleotides literally, TATA repeats. Transcription factors bind to the TATA box, assembling an initiation complex.
Once this complex is assembled, RNA polymerase binds to its upstream sequence and becomes phosphorylated. This releases part of the protein from the DNA, activates the transcription initiation complex, and places RNA polymerase in the correct orientation to begin transcription Figure In some eukaryotic genes, there are regions that help increase transcription. These regions, called enhancers , are not necessarily close to the genes; they can be located thousands of nucleotides away.
They can be found upstream, within the coding region, or downstream of a gene. Enhancers are binding sites for activators. When an enhancer is far away from a gene, the DNA folds such that the enhancer is brought into proximity with the promoter, allowing interaction between the activators and the transcription initiation complex Figure Like prokaryotic cells, eukaryotic cells also have mechanisms to prevent transcription.
Transcriptional repressors can bind to promoter or enhancer regions and block transcription. Both activators and repressors respond to external stimuli to determine which genes need to be expressed. Post-transcriptional regulation occurs after the mRNA is transcribed but before translation begins.
This regulation can occur at the level of mRNA processing, transport from the nucleus to the cytoplasm, or binding to ribosomes.
Recall from chapter 5 that in eukaryotic cells the RNA primary transcript often contains introns, which are removed prior to translation. Alternative RNA splicing is a mechanism that allows different combinations of introns, and sometimes exons, to be removed from the primary transcript Figure This allows different protein products to be produced from one gene.
Alternative splicing can act as a mechanism of gene regulation. Differential splicing is used to produce different protein products in different cells or at different times within the same cell. Alternative splicing is now understood to be a common mechanism of gene regulation in eukaryotes; up to 70 percent of genes in humans are expressed as multiple proteins through alternative splicing.
How could alternative splicing evolve? Introns have a beginning and ending recognition sequence; it is easy to imagine the failure of the splicing mechanism to identify the end of an intron and instead find the end of the next intron, thus removing two introns and the intervening exon.
In fact, there are mechanisms in place to prevent such intron skipping, but mutations are likely to lead to their failure. Indeed, the cause of many genetic diseases is alternative splicing rather than mutations in a sequence. However, alternative splicing would create a protein variant without the loss of the original protein, opening up possibilities for adaptation of the new variant to new functions. Gene duplication has played an important role in the evolution of new functions in a similar way by providing genes that may evolve without eliminating the original, functional protein.
The longer an mRNA exists in the cytoplasm, the more time it has to be translated, and the more protein is made. Many factors contribute to mRNA stability, including the length of its poly-A tail. After an mRNA has been transported to the cytoplasm, it is translated into proteins. Control of this process is largely dependent on the mRNA molecule. As previously discussed, the stability of the mRNA will have a large impact on its translation into a protein. Translation can also be regulated at the level of binding of the mRNA to the ribosome.
Once the mRNA bound to the ribosome, the speed and level of translation can still be controlled. An example of translational control occurs in proteins that are destined to end up in an organelle called the endoplasmic reticulum ER. The first few amino acids of these proteins are a tag called a signal sequence.
As soon as these amino acids are translated, a signal recognition particle SRP binds to the signal sequence and stops translation while the mRNA-ribosome complex is shuttled to the ER. Once they arrive, the SRP is removed and translation resumes. The final level of control of gene expression in eukaryotes is post-translational regulation. This type of control involves modifying the protein after it is made, in such as way as to affect its activity.
One example of post-translational regulation is enzyme inhibition. When an enzyme is no longer needed, it is inhibited by a competitive or allosteric inhibitor, which prevents it from binding to its substrate. The inhibition is reversible, so that the enzyme can be reactivated later. This is more efficient than degrading the enzyme when it is not needed and then making more when it is needed again. Sometimes these modifications can regulate where a protein is found in the cell—for example, in the nucleus, the cytoplasm, or attached to the plasma membrane.
The addition of an ubiquitin group to a protein marks that protein for degradation. Tagged proteins are moved to a proteasome , an organelle that degrades proteins Figure Several other introns are like AG intron 2 in that they are required to restrict gene expression in some way, and may be considered negative enhancer elements.
Similarly, deletions within the first intron of the wheat VRN-1 gene are associated with reduced repression of this gene in response to vernalization Fu et al. The AG, STK, FLC, and VRN-1 introns are all large, which may provide sufficient room for numerous controlling elements or permit the establishment of a local chromatin conformation required for appropriate expression. Alternative Promoters A few introns are capable of driving at least weak expression of a promoterless gene and therefore contain promoters that could be partly responsible for increasing expression.
Rose the rice introns mentioned contain alternative promoters is supported by evidence that some transcripts originate within intron sequences. The functional significance of the intronic promoter is unclear, as both forms of mRNA encode identical proteins and transcripts starting in the intron are far less abundant. Intron-Mediated Enhancement Not long after introns were first shown to elevate expression in mammalian cells, Callis et al. This work is remarkable for its thoroughness and the number of features of IME for which this paper is the first published record in plants.
These characteristics have become the defining features of IME and made it clear that the introns studied were operating by a mechanism unrelated to conven- tional enhancer elements.
Monocots and Dicots The earliest experiments on IME in plants were done in maize, in which several introns were shown to stimulate expression up to fold Callis et al. In contrast, some of the first dicot genes to be tested are expressed at similar or possibly higher levels without their introns Chee et al.
This led to the widespread belief that IME is more robust in monocots than dicots. Since then, claims of dicot introns that increase expression 1,fold or more have been published Curie et al. What appeared to be a trend may simply reflect differences in the enhancing ability of the introns that happened to be chosen first for study.
As more introns are analyzed, the differ- ence in the magnitude of IME in monocots and dicots is becoming less obvious. The approach of testing the same intron in both monocot and dicot species has given mixed results Last et al. The retention of any intron in a transcript would interfere with expression and possibly destabilize the mRNA through NMD.
Thus a direct comparison of IME in monocots and dicots by the same intron may not reveal true differences between the groups unless effi- cient splicing in both is demonstrated. Also, the species specificity of the IME machinery remains unknown, although some introns can clearly enhance expres- sion in many widely divergent plants.
A potentially significant difference between the published descriptions of IME in monocots and dicots is that virtually all monocot studies have been done with transient expression assays, while many dicot studies have used stably transformed plants, which are easier to generate than in monocots.
IME has been observed in transient assays in dicots, although in studies in which both methods could be com- pared the effect of the intron was more pronounced in the stable transformants Jeong et al. It will be interesting to see whether the mag- nitude of IME is greater in stably transformed monocots than in transient assays, as appears to be the case in rice Tanaka et al.
Sequences Involved in IME Further evidence that many introns stimulate mRNA levels without containing dis- crete enhancers or promoters came from attempts to define the intron sequences involved in IME through deletion analysis. Similarly, deleting nt from the middle of the nt first intron from the maize Sh1 gene has little effect on the ability of this intron to stimulate expression Clancy and Hannah ; Clancy et al.
Deleting of the nt that comprise the rice OstubA1 first intron causes the enhancement to decline, although the remaining nt derivative still enhances roughly twentyfold Jeon et al. For these introns, most or all of the sequences responsible for IME must be redundant and distributed throughout the intron, or are limited to the ends of the intron.
One consequence of the inability of deletions to abolish IME is that no specific sequence has been shown to be absolutely required, and even the general nature of the sequences involved remains unclear. No motifs conserved between enhancing introns have been found. Deletions within a nt truncated version of the maize Sh1 first intron identified a 35 nt region whose removal reduces enhancement by half from fold to approximately fold.
Full enhancement can be restored by substituting this region with a similarly U-rich region from another part of the intron Clancy and Hannah In contrast to these U-rich candidates, a GC-rich octamer from the maize GapA1 first intron was tenta- tively identified as an enhancing sequence Donath et al. Our understanding of the mechanism of IME would be greatly advanced by a clear definition of the intron sequences responsible for stimulating expression.
Early tests of the need for splicing in IME found that mutations that prevented splicing also eliminated the enhancing effect of an intron Sinibaldi and Mettler This suggests that the mechanism of IME requires that the splicing machinery be at least partly assembled onto an intron, even if it is unable to complete its task. Introns might boost expression simply by providing an association between a transcript and the splicing machinery that increases the efficiency of other steps of pre-mRNA maturation, leading to greater mRNA production.
The many reactions of gene expression are known to be interconnected and affect each other Maniatis and Reed If interactions between pre-mRNA and the spliceosome are the sole basis of IME, then all efficiently spliced introns should enhance equally well.
Unfortunately, it has proven difficult to place an absolute or even a relative value on the stimulating ability of different introns. The magnitude of enhancement usu- ally cannot be compared between publications because IME is influenced by many factors other than the intron, including the promoter and coding sequences of the gene used to monitor expression Callis et al.
Even within a single study, the intron may not be the only difference between constructs because the engineering used to insert an intron often creates additional changes in the encoded protein or mature mRNA.
While usually minor, these sequence differences could affect mRNA stabil- ity, translation, or the activity of the reporter enzyme.
Some genes essentially require introns in order to be expressed at all, precluding an accurate determination of the fold change in expression caused by an intron. In other cases, introns alter the tissue- specific expression pattern of a gene Casas-Mollano et al. Despite these caveats, studies in which variables have been minimized clearly show that introns vary widely in their ability to stimulate expression.
Each of the six is efficiently spliced, and the resulting mature mRNA is identical in all cases. The degree to which mRNA accumulation is increased ranges from less than twofold for the first intron from the TCH3 gene to more than fold for introns from UBQ10 or atpk1.
In maize, the effects of seven introns on transient expression range from negligible to more than fold, depending on the intron and reporter gene Sinibaldi and Mettler The observation that some efficiently spliced introns fail to enhance means that simply providing an association between a pre-mRNA and the splicing machinery is not sufficient to cause IME, and suggests that the degree to which an intron stim- ulates expression is determined by elements other than the highly conserved sequences involved in the splicing reactions.
Intron Position Investigations into the role of intron location have furnished a potentially signifi- cant clue about the mechanism of IME. The position of an intron can influence its ability to enhance expression in two ways.
The first is the location of the intron within the gene from which it was isolated. The other way in which position is important is that introns must be located near the start of a gene for maximal enhancement.
Rose While introns elevate mRNA accumulation, they apparently manage to do this without increasing either the rate of transcription or mRNA stability. Introns that significantly increase mRNA accumulation have at most a minor effect on the signal generated in nuclear run-on transcription assays Dean et al.
Similarly, the stability of mRNAs derived from intron-containing and intronless genes are indistinguishable Nash and Walbot ; Rethmeier et al. Comparable results have been obtained in mammalian cells Lu and Cullen ; Nott et al.
How can these apparently incompatible results be reconciled? One possibility is that the run-on assays yield misleading results because delicate or transient intron-mediated changes in the transcription machin- ery could be lost during the isolation of the nuclei used in the assays. Another is that while run-on transcription assays measure mostly initiation, introns might pri- marily affect transcript elongation.
While both could be true, the latter possibility is most consistent with the need for introns to be near the start of a gene for maxi- mum effect, and forms the basis for the following model. In the absence of introns, transcription would initiate at the same rate but the polymerase may be more likely to stall or fall off the template, leaving an abortive transcript that is rapidly degraded. Full-length transcripts from intronless genes could still be made and would be just as stable but would be less abundant.
How might introns be able to affect the transcription machinery? Intron sequences in the DNA might adopt a particular configuration or nucleosome association that eases passage of the transcription machinery or makes it more processive in some way.
If the higher-order structure of the DNA makes introns easier to tran- scribe, they might be expected to function in either orientation because of the double-stranded nature of DNA, but this is not the case. Introns must be in the proper orientation for IME.
However, the conserved splice site nucleotides and U-richness of most introns are not preserved in the other orientation, making it very unlikely that the backwards intron would be recognized as an intron and properly spliced. The need for splicing, or at least an association of the intron with the splicing machinery, is perhaps the strongest evidence that IME acts at the RNA level.
In this case, some redundant and dispersed sequences in a newly transcribed intron might bind to factors that in turn communicate with the transcription machinery to render it more processive. Transcription is regulated by changes in the phosphorylation of certain residues in the repeats that constitute the CTD, and splicing factors are known to interact with the CTD in a phosphorylation-dependent manner Steinmetz This established link between introns and PolII provides a plausible opportunity for introns to affect the phospho- rylation of the CTD and thus the activity of the transcription machinery.
The crucial link-factors that bind to introns and modify PolII-currently remains missing. Identifying dispersed sequences that are involved in IME may provide a means to isolate such factors if they exist. Regardless of whether introns act at the DNA or RNA level, an effect on tran- script elongation would make biological sense if introns are one of the ways in which the transcription machinery can differentiate between genes and intergenic spaces.
Transcripts that initiate spontaneously at sequences that are not genuine promoters or fail to terminate at the end of real genes are potentially dangerous because they can form antisense transcripts that could interfere with the expression of other genes.
This risk would be reduced if the transcription machinery had the highest processivity only in actual genes, which could be recognized by the pres- ence of introns.
Conclusions It is not surprising that many different types of gene regulation involve introns. Introns are largely free of the sequence constraints inherent in coding sequences and therefore present many opportunities for the rapid evolution of new elements that control gene expression. Enough different kinds of regulation have been characterized to illustrate the danger in assuming that all introns affect expres- sion by the same mechanism.
The mysterious nature of IME suggests that introns still have a lot to teach us about how eukaryotic gene expression is regulated.
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