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Cis-Acting Proteins
Cis-Acting Proteins KM Derbyshire, State University of New York at Albany, Albany, NY, USA
© 2001 Elsevier Inc. All rights reserved.
This article is reproduced from the previous edition, volume 1, pp 380–382, © 2001, Elsevier Inc.
Cis-Acting proteins are an unusual class of DNA-binding pro teins that act preferentially on DNA sites located close to the gene from which they are expressed. This is in sharp contrast to the majority of proteins, which are freely diffusible (trans acting) and can act at many different locations in the genome with equal efficiency. In fact, a protein’s ability to freely diffuse in a bacterium is a basic requirement of the classical comple mentation test used to determine if two mutations affect the same gene function. Cis-Acting proteins were originally identi fied using such an assay: they exhibited weak complementation of a defective allele when a wild-type copy of the gene was supplied in trans.
Classification of Cis-Acting Proteins Most examples of cis-acting proteins have been described in bacteria where the coupling of transcription and translation ensures that a protein is synthesized in the vicinity of its gene, which fulfills one of the requirements for cis action (see below). The discrete compartmentalization of transcription and translation in eukaryotes prevents such a localized synth esis. In fact, the few examples of cis preference described in higher organisms have involved proteins acting in cis on their mRNA. Bacterial cis-acting proteins have been divided into three groups based on their function; however, proteins within a class do not achieve cis action by the same mechanism. The largest and most well-studied class of cis-acting proteins con sists of the transposases, encoded by bacterial insertion elements. Proteins associated with replication of certain single-stranded phage (e.g., the CisA protein of phi X174) and bacterial plasmids (the RepA protein of plasmid R1) form the second group of cis-acting proteins. The third class includes regulatory proteins such as the bacteriophage lambda antitermination protein, Q, and the D-serine deaminase activator protein of Escherichia coli.
What Purpose Does Cis Action Serve? All the cis-acting proteins described to date play a critical role in DNA/RNA metabolism or regulation wherein restriction of activation to a single genetic unit is beneficial to the survival of the cell and/or the genetic element encoding the protein. This is especially true of the insertion sequence (IS) transpo sases. Bacterial IS elements transpose predominantly by a cut and-paste (donor-suicide mechanism) mechanism that leaves potentially lethal double-strand breaks in the chromosome. These transposons are often found in multiple copies within a cell and many of these are defective due to the acquisition of deleterious mutations. High expression of a trans-acting trans posase, encoded by a cut-and-paste transposon, would result in large-scale activation of cryptic elements within a genome and
12
lead to many deleterious IS insertions and DNA rearrange ments. Thus, cis action limits transposition to a single element, ensures that distant defective elements are not acti vated, and provides a selective process to enrich for active transposons. Interestingly, transposons that move via a repli cative mechanism that does not involve double-strand breaks encode trans-acting transposases.
How Is Cis Action Achieved? To explain cis preference, most models propose that there is an unequal distribution of protein within the cell such that the highest concentration of active protein exists around its site of action – close to the gene encoding the protein. To generate such a gradient requires that (1) protein synthesis be limited to the immediate vicinity of the gene and (2) dif fusion of the protein to other sites in the genome be restricted. How this gradient is achieved and maintained has been the focus of much research and, not surprisingly, is accomplished in several distinct ways. The most signifi cant insight into these mechanisms has been gained by the isolation and characterization of protein mutants, or the development of conditions, that allow a cis-acting protein to become trans-acting.
Localizing Protein Synthesis to the Vicinity of Its Gene In bacteria, the natural coupling of transcription and transla tion results in localized protein synthesis. Consequently, any process that enhances this coupling will increase the likelihood of action in cis. For example, slow release of mRNA from its DNA template will increase the tethering of the mRNA (and therefore protein) to its gene. The rate of degradation of a transcript will also influence the location of protein synthesis: a long mRNA half-life would allow time for the message to diffuse away from the gene and therefore facilitate trans action. Examples of such regulation have been described for the IS10 transposase. The cis preference of the transposase is influ enced by mutations that affect the release and stability of the transposase mRNA. Mutations that increase the rate of transla tional initiation result in an increased rate of transcript release and also an increased half-life of the mRNA, as ribosomes protect the mRNA from nucleases. The net effect of this is to increase the amount of diffusible transcript, resulting in a more even distribution of protein in the cell. The cis action of the RepA protein of the plasmid R1 has also been attributed to transcriptional tethering. This protein is required for initiation of plasmid replication. A Rho-dependent transcription termination site located at the 3′ end of the repA gene is thought to cause the RNA polymerase complex to stall, thereby increasing the length of time the mRNA is tethered to its template and thus
Brenner’s Encyclopedia of Genetics, 2nd Edition, Volume 2
doi:10.1016/B978-0-12-374984-0.00255-2
Cis-Acting Proteins facilitating the delivery of RepA protein to sites associated with the repA gene. An unusual form of cis preference, also based on tethering, has been proposed to explain the lack of complementation observed between the multiple copies of LINE (L1) retrotran sposons found in mammals. Although these elements are extremely abundant, only a small fraction of them actually transpose. It is thought that the nature of retrotransposition, which occurs via an RNA intermediate, plays a key role in cis action. The L1 transcript has two roles. It is the template for translation of two proteins required for transposition. One of these, the ORF2 protein, encodes reverse transcriptase and endonuclease activities and is thought to bind to the polyA tail of its own mRNA immediately following translation. The transcript also acts as the template for target-primed reverse transcription mediated by ORF2. Thus, ORF2 preferentially reverse transcribes its own mRNA in the transposition process.
Mechanisms That Limit Protein Diffusion and Thereby Enhance Cis Preference To maintain the gradient established by coupled transcription and translation, it is important to limit the redistribution of protein to other locations in the genome. There is a variety of processes that serve to accomplish this and in many cases more than one is employed to reduce trans action.
Protein Instability Cis Preference of the IS903 transposase has been correlated with its very short half-life. Mutations of transposase, or conditions that increase protein stability, increase its ability to comple ment defective transposons in trans by allowing more time for the protein to diffuse through the cell before it is inactivated. This requires that the protein be made in limiting amounts and that the time taken to find a distant site (in trans) must be longer than the half-life of the protein (see section Sequestration of Protein). In fact, the IS903 transposase, like many other IS transposases, is poorly expressed. Since transpo sition is thought to require multimers of transposase, limiting the amount of protein synthesized will also reduce the like lihood that the concentration of protein at trans sites would be sufficient to form the multimers that catalyze transposition.
13
multimerization of the transposase reduces the functional half-life of non-DNA-bound protein.
Multiple Binding Sites A third way to reduce the redistribution of protein is to have multiple binding sites for the protein in the vicinity of the gene. This situation is observed with the repA gene of the plasmid R1, which is closely linked to a multiple array of RepA-binding sites thought to trap the protein and prevent further redistribution. An extension of this type of model is simply to propose that the protein in question has a relatively high affinity for nonspecific DNA compared with its specific DNA-binding site. In this scenario, the protein would spend extended periods of time associated with nonspecific DNA, which would contribute to cis preference by slowing diffusion away from its site of synthesis.
More Cis-Acting Proteins? To date, only extreme cases of cis preference have been docu mented. As other regulatory systems are characterized and genetic systems developed in other organisms, it is likely that other examples of cis-acting proteins will be described, but perhaps with more subtle cis preferences (i.e., an intermediate phenotype). Given the different schemes that have been iden tified to date, it would not be surprising if cis action can be achieved by yet other novel processes. Further examples of cis-acting proteins are likely to be described in eukaryotes as new and improved genetic systems allow more precise moni toring of complementation analyses. The example of LINE elements certainly suggests that other retrotransposons may encode proteins that preferentially act on their RNA templates. Mobile group II introns move by a similar mechanism to LINE-like elements, and thus might also be expected to favor insertion of a copy of the RNA template from which its proteins were encoded. Preliminary evidence indicates that a cis-acting protein(s) may be required for replication of the RNA-based poliovirus and thus suggests that RNA viruses might also be a new, untapped source of cis-acting proteins. By extension, other RNA-mediated processes may also utilize cis preference for regulation.
See also: Complementation Test; Insertion Sequences; Retrotransposons.
Sequestration of Protein Reducing the functional half-life of the protein by increasing the time required to find a distant site can be achieved by sequestering the protein. The CisA replication initiation protein of phi X174 is quickly sequestered in the membrane away from its site of action in the A gene. Thus, the membrane acts as a trap, reducing the availability of protein to other genomic sites. Formation of inactive multimers is thought to sequester the IS50 transposase away from trans sites and favor cis action of the protein. A derivative of IS50 transposase that reduces dimerization with either itself or a transposase inhibitor pro tein increases trans activity, suggesting that nonproductive
Further Reading Derbyshire KM and Grindley NDF (1996) Cis-preference of the IS903 transposase is mediated by a combination of transposase instability and inefficient translation. Molecular Microbiology 21: 1261–1272. Jain C and Kleckner N (1993) Preferential cis action of IS10 transposase depends upon its mode of synthesis. Molecular Microbiology 9: 249–260. Kazazian HH and Moran JV (1998) The impact of L1 retrotransposons on the human genome. Nature Genetics 19: 19–24. Novak JE and Kirkegaard K (1994) Coupling between genome translation and replication in an RNA virus. Genes & Development 8: 1726–1737. Wei W, Gilbert N, Coi SL, et al. (2001) Human L1 retrotransposition: cis Preference versus trans complementation. Molecular and Cellular Biology 21: 1429–1439.
© 2001 Elsevier Inc. All rights reserved.
This article is reproduced from the previous edition, volume 1, pp 380–382, © 2001, Elsevier Inc.
Cis-Acting proteins are an unusual class of DNA-binding pro teins that act preferentially on DNA sites located close to the gene from which they are expressed. This is in sharp contrast to the majority of proteins, which are freely diffusible (trans acting) and can act at many different locations in the genome with equal efficiency. In fact, a protein’s ability to freely diffuse in a bacterium is a basic requirement of the classical comple mentation test used to determine if two mutations affect the same gene function. Cis-Acting proteins were originally identi fied using such an assay: they exhibited weak complementation of a defective allele when a wild-type copy of the gene was supplied in trans.
Classification of Cis-Acting Proteins Most examples of cis-acting proteins have been described in bacteria where the coupling of transcription and translation ensures that a protein is synthesized in the vicinity of its gene, which fulfills one of the requirements for cis action (see below). The discrete compartmentalization of transcription and translation in eukaryotes prevents such a localized synth esis. In fact, the few examples of cis preference described in higher organisms have involved proteins acting in cis on their mRNA. Bacterial cis-acting proteins have been divided into three groups based on their function; however, proteins within a class do not achieve cis action by the same mechanism. The largest and most well-studied class of cis-acting proteins con sists of the transposases, encoded by bacterial insertion elements. Proteins associated with replication of certain single-stranded phage (e.g., the CisA protein of phi X174) and bacterial plasmids (the RepA protein of plasmid R1) form the second group of cis-acting proteins. The third class includes regulatory proteins such as the bacteriophage lambda antitermination protein, Q, and the D-serine deaminase activator protein of Escherichia coli.
What Purpose Does Cis Action Serve? All the cis-acting proteins described to date play a critical role in DNA/RNA metabolism or regulation wherein restriction of activation to a single genetic unit is beneficial to the survival of the cell and/or the genetic element encoding the protein. This is especially true of the insertion sequence (IS) transpo sases. Bacterial IS elements transpose predominantly by a cut and-paste (donor-suicide mechanism) mechanism that leaves potentially lethal double-strand breaks in the chromosome. These transposons are often found in multiple copies within a cell and many of these are defective due to the acquisition of deleterious mutations. High expression of a trans-acting trans posase, encoded by a cut-and-paste transposon, would result in large-scale activation of cryptic elements within a genome and
12
lead to many deleterious IS insertions and DNA rearrange ments. Thus, cis action limits transposition to a single element, ensures that distant defective elements are not acti vated, and provides a selective process to enrich for active transposons. Interestingly, transposons that move via a repli cative mechanism that does not involve double-strand breaks encode trans-acting transposases.
How Is Cis Action Achieved? To explain cis preference, most models propose that there is an unequal distribution of protein within the cell such that the highest concentration of active protein exists around its site of action – close to the gene encoding the protein. To generate such a gradient requires that (1) protein synthesis be limited to the immediate vicinity of the gene and (2) dif fusion of the protein to other sites in the genome be restricted. How this gradient is achieved and maintained has been the focus of much research and, not surprisingly, is accomplished in several distinct ways. The most signifi cant insight into these mechanisms has been gained by the isolation and characterization of protein mutants, or the development of conditions, that allow a cis-acting protein to become trans-acting.
Localizing Protein Synthesis to the Vicinity of Its Gene In bacteria, the natural coupling of transcription and transla tion results in localized protein synthesis. Consequently, any process that enhances this coupling will increase the likelihood of action in cis. For example, slow release of mRNA from its DNA template will increase the tethering of the mRNA (and therefore protein) to its gene. The rate of degradation of a transcript will also influence the location of protein synthesis: a long mRNA half-life would allow time for the message to diffuse away from the gene and therefore facilitate trans action. Examples of such regulation have been described for the IS10 transposase. The cis preference of the transposase is influ enced by mutations that affect the release and stability of the transposase mRNA. Mutations that increase the rate of transla tional initiation result in an increased rate of transcript release and also an increased half-life of the mRNA, as ribosomes protect the mRNA from nucleases. The net effect of this is to increase the amount of diffusible transcript, resulting in a more even distribution of protein in the cell. The cis action of the RepA protein of the plasmid R1 has also been attributed to transcriptional tethering. This protein is required for initiation of plasmid replication. A Rho-dependent transcription termination site located at the 3′ end of the repA gene is thought to cause the RNA polymerase complex to stall, thereby increasing the length of time the mRNA is tethered to its template and thus
Brenner’s Encyclopedia of Genetics, 2nd Edition, Volume 2
doi:10.1016/B978-0-12-374984-0.00255-2
Cis-Acting Proteins facilitating the delivery of RepA protein to sites associated with the repA gene. An unusual form of cis preference, also based on tethering, has been proposed to explain the lack of complementation observed between the multiple copies of LINE (L1) retrotran sposons found in mammals. Although these elements are extremely abundant, only a small fraction of them actually transpose. It is thought that the nature of retrotransposition, which occurs via an RNA intermediate, plays a key role in cis action. The L1 transcript has two roles. It is the template for translation of two proteins required for transposition. One of these, the ORF2 protein, encodes reverse transcriptase and endonuclease activities and is thought to bind to the polyA tail of its own mRNA immediately following translation. The transcript also acts as the template for target-primed reverse transcription mediated by ORF2. Thus, ORF2 preferentially reverse transcribes its own mRNA in the transposition process.
Mechanisms That Limit Protein Diffusion and Thereby Enhance Cis Preference To maintain the gradient established by coupled transcription and translation, it is important to limit the redistribution of protein to other locations in the genome. There is a variety of processes that serve to accomplish this and in many cases more than one is employed to reduce trans action.
Protein Instability Cis Preference of the IS903 transposase has been correlated with its very short half-life. Mutations of transposase, or conditions that increase protein stability, increase its ability to comple ment defective transposons in trans by allowing more time for the protein to diffuse through the cell before it is inactivated. This requires that the protein be made in limiting amounts and that the time taken to find a distant site (in trans) must be longer than the half-life of the protein (see section Sequestration of Protein). In fact, the IS903 transposase, like many other IS transposases, is poorly expressed. Since transpo sition is thought to require multimers of transposase, limiting the amount of protein synthesized will also reduce the like lihood that the concentration of protein at trans sites would be sufficient to form the multimers that catalyze transposition.
13
multimerization of the transposase reduces the functional half-life of non-DNA-bound protein.
Multiple Binding Sites A third way to reduce the redistribution of protein is to have multiple binding sites for the protein in the vicinity of the gene. This situation is observed with the repA gene of the plasmid R1, which is closely linked to a multiple array of RepA-binding sites thought to trap the protein and prevent further redistribution. An extension of this type of model is simply to propose that the protein in question has a relatively high affinity for nonspecific DNA compared with its specific DNA-binding site. In this scenario, the protein would spend extended periods of time associated with nonspecific DNA, which would contribute to cis preference by slowing diffusion away from its site of synthesis.
More Cis-Acting Proteins? To date, only extreme cases of cis preference have been docu mented. As other regulatory systems are characterized and genetic systems developed in other organisms, it is likely that other examples of cis-acting proteins will be described, but perhaps with more subtle cis preferences (i.e., an intermediate phenotype). Given the different schemes that have been iden tified to date, it would not be surprising if cis action can be achieved by yet other novel processes. Further examples of cis-acting proteins are likely to be described in eukaryotes as new and improved genetic systems allow more precise moni toring of complementation analyses. The example of LINE elements certainly suggests that other retrotransposons may encode proteins that preferentially act on their RNA templates. Mobile group II introns move by a similar mechanism to LINE-like elements, and thus might also be expected to favor insertion of a copy of the RNA template from which its proteins were encoded. Preliminary evidence indicates that a cis-acting protein(s) may be required for replication of the RNA-based poliovirus and thus suggests that RNA viruses might also be a new, untapped source of cis-acting proteins. By extension, other RNA-mediated processes may also utilize cis preference for regulation.
See also: Complementation Test; Insertion Sequences; Retrotransposons.
Sequestration of Protein Reducing the functional half-life of the protein by increasing the time required to find a distant site can be achieved by sequestering the protein. The CisA replication initiation protein of phi X174 is quickly sequestered in the membrane away from its site of action in the A gene. Thus, the membrane acts as a trap, reducing the availability of protein to other genomic sites. Formation of inactive multimers is thought to sequester the IS50 transposase away from trans sites and favor cis action of the protein. A derivative of IS50 transposase that reduces dimerization with either itself or a transposase inhibitor pro tein increases trans activity, suggesting that nonproductive
Further Reading Derbyshire KM and Grindley NDF (1996) Cis-preference of the IS903 transposase is mediated by a combination of transposase instability and inefficient translation. Molecular Microbiology 21: 1261–1272. Jain C and Kleckner N (1993) Preferential cis action of IS10 transposase depends upon its mode of synthesis. Molecular Microbiology 9: 249–260. Kazazian HH and Moran JV (1998) The impact of L1 retrotransposons on the human genome. Nature Genetics 19: 19–24. Novak JE and Kirkegaard K (1994) Coupling between genome translation and replication in an RNA virus. Genes & Development 8: 1726–1737. Wei W, Gilbert N, Coi SL, et al. (2001) Human L1 retrotransposition: cis Preference versus trans complementation. Molecular and Cellular Biology 21: 1429–1439.