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Evolution of the rDNA multigene family
14
S37
Cell Biology International
NUCLEOLUS STRUCTURE
AND
FUNCTION
Ulrich Scheer and Ricardo Benavente. Institute of Zoology, University of Wiirzburg, D8700 Wiirzburg, Federal Republic of Germany. Nucleoli offer the possibility of correlating specific steps of ribosome biosynthesis with structurally distinct components. In particular, the location of the active rRNA genes is essential towards a full understanding of nucleolus structure and function. Our EM immunogold labelling studies with antibodies to RNA polymerase I (RPI), topoisomerase I and DNA point to the fibrillar centers (FC) as the exclusive sites of the transcribing rRNA genes. Comparison of the dimensions of the rRNA transcription units with those of the FCs reveals an extremely high packing density of the active genes in situ which might provide a mechanism for creating high local concentrations of RPI and transcription factors in the FCs. Inhibition of rDNA transcription by microinjection of antibodies to RPI or exposure of cells to campthotecin leads to a disintegration of nucleoli. Maintainance of their structural integrity as well as their de novo assembly mitosis following requires the activity of the rRNA genes. Nascent transcripts seem to key role in these processes. Since play a we could locate fibrillarin, a protein constituent of U3 RNP, to the free ends of growing transcripts we propose that the 5'regions of nascent pre-rRNA molecules link the active genes of the FCs to the surrounU. and ding nucleolar components (Scheer, Benavente, R., BioEssays 12, 14-21, 1990).
MOLECULAR MECHANISMS REGULATING RISOSOMAL GENE TRANSCRIPTION IN MAMMALIAN CELLS lngrid Grummt, Andraas Schnapp and Anne Kuhn. Institute of Biochemistry Rlrntgenring 11. 8700 WOnburg, F.R.G. Mouse RNA polymerase I transcription involves at least three auxiliary transcription initiation factors, termed TIF-IA. TIFIB and TIF-IC. Factor TIF-IB has been purified to molecular homogeneity. It is a 44 kd DNA binding protein which specifically interacts with he core region (from -39 to -1) of the rDNA promoter and confers promoter specificity to RNA polymerase I. Control of rRNA synthesis in response to the physiological state of the calls is brought about by TIF-IA, a regulatory factor whose amount or activity correlates with cell proliferation. This growth-dependent factor TIF-IA interacts with RNA polymerase I, thus converting it into a transcriptionally active holoenzyme, which is able to initiate specifically at the rDNA promoter in the presence of the other transcription factors, TIF-IB and TIF-IC. In addition, regulation of rRNA synthesis appears to involve a specific posttranslational modification of RNA polymerase I which is required for transcription initiation. Treatment of RNA polymerase I with alkaline phosphatase abolishes specific initiation at the rDNA promoter without significantly affecting the polymerizing activity of the enzyme at nonspecific templates. The data propose an efficient and versatile mechanism of rDNA transcription regulation which enables the cell to quickly adapt ribosome biosynthesis lo a variety of extracellular signals. Furthermore, we show that the 140 nt repetitive sequence elements located upstream from the gene promoter function as polymerase l-specific transcription enhancers. These repeats stimulate transcription of an adjacent gene promoter in cis and compete for transcription in trans. indicating that the enhancer elements bind at least one common factor which is required for promoter activity.
s39
Reports, Vol. 14, Abstracts Supplement
S38
EVOLUTION
OF THE
rDNA
MULTIGENE
1990
FAMILY
John M. Hancock & Gabriel A. Dover, Dept. of Genetics, University of
Cambridge, Downing Street, CambridgeCB23EH,U.K. The rDNA of eukaryotesis a mu&gene family in which a high level of similarity exists betwwn copiesof its basic repeat unit, primarily becauseof the homogeneizingconsequencesof unequalcrossingover. How are essential rDNA functions maintained during homogeneization? Three examples of “molecular ccevolution” reveal how this might be achieved. 1. Promoter-Polymerase
Coevolution
An interspecific incompatibility often exists between rDNA promoters and RNA polymerase I complexes. Recent evidences in humans, wheat and Drosophila suggest that this arises through coevolution between a newly homogenized promoter variant and a polymerase I cofactor, and between different cofactors. 2. Expansion
Segment
Coevolution
The expansion segmentsof large subunit rRNAs coevolve in many species by the acquisition of similar short, interspersed sequence motifs, probably generated by DNA slippage. The set of epansion segments in one species share motifs that are generally distinct from those in another species. 3. Compensatory Mutation and Compensatory Slippage When a point mutation occurs within a secondary structural stem of an rRNA molecule, it is often compensated by a second mutations which restores the base pairing in that position. Similarly, repetitive motifs are incorporated in
such a way that pre-existing stems are increased in length by a process of compensatory slippage. These processes are difficult to understand in the context of a multigene family but may be explained if the family can tolerate the spread of an uncompensatedmutation before compensation ties place. References:
Dover, G.A. (1982) “Molecular drive. (I cohesive mode of species evolution” Nature 299: 111-117: Dover. G.A. & Flavell. R.H. (1984) “Molecular coevolution: DNA divergence &d the maintenance .of fur&m” Ckll 38: 622-623: Hancock.J.M. & Dover G.A. (1988) “Molecular coevolurion among cryptically simple expansion segmenrsof eukaryoric 26Sl28S rRNAs” Mol. Biol. Evol. 5: 377.391; Hancock,J.M. et al. (1988) “Evolution of the secondary SWUC~WPS and compensntory mutations of the ribosomol RNAs of Drosophila melanogaster” slippage Mol. Biol. Evol. 5: 393-414:Hancock& Dover (1990) “Compensatory in the evolurion of ribosomol RNA genes” Science. sub&led.
COORDINATE CONTROL OF RJBOSOMAL PROTEIN SYNTHRS ISINYRAST Rudi J. Planta, Willem H. Mager, Thijs M. Doorenbosch and Leon S. Kraakman. Biochemisch Laboratorium, Vrije Universiteit, de Boelelaan 1083, Amstuxlam. The Netherlands.
s40
Ribosomal protein synthesis @p-synthesis) in yeast is a coordinate process resulting in the equimolar production of ahout 80 different ribosomal proteins. The rate of q-synthesis is precisely adjusted to the celhzlar growth rate. Regulation of rp-synthesis oeews primuily at the transcriptional level. Transcription activation of the majority of the ‘p-genes is mediated through common upstream activating sequences. socalled RPO-boxes, which usually are located at a distance of 200-500 tap from the start cadon. These RFSboxes represent hiding sites for a transaiptional activator, called TUP or RAP. The concenuation of TUF parallels the cellular growth rate and eviti exists that the reqmsc in expression of q-genes upon nutritional changes is medkted by this factor. Recent findings indicate that TUP/RAP also activates other gene families iuvokd in dhlar growth rate. In add&ion, this multifunctional pOthlhhdStOthC~-~-andtC-iUyeast. Some other rp-gum (e.g. those cnmding S33 and L45) do notcontainanRpG-hox.Theyappeartobeactivatcdhyanother multifunctional protein, called ABFl or SUP, which hinds to a specific squence element. This multifimctional protein also aetivatesolhcrlgMef,audhladditionhindstothemathlg type silencer ad ARSclrments.
S37
Cell Biology International
NUCLEOLUS STRUCTURE
AND
FUNCTION
Ulrich Scheer and Ricardo Benavente. Institute of Zoology, University of Wiirzburg, D8700 Wiirzburg, Federal Republic of Germany. Nucleoli offer the possibility of correlating specific steps of ribosome biosynthesis with structurally distinct components. In particular, the location of the active rRNA genes is essential towards a full understanding of nucleolus structure and function. Our EM immunogold labelling studies with antibodies to RNA polymerase I (RPI), topoisomerase I and DNA point to the fibrillar centers (FC) as the exclusive sites of the transcribing rRNA genes. Comparison of the dimensions of the rRNA transcription units with those of the FCs reveals an extremely high packing density of the active genes in situ which might provide a mechanism for creating high local concentrations of RPI and transcription factors in the FCs. Inhibition of rDNA transcription by microinjection of antibodies to RPI or exposure of cells to campthotecin leads to a disintegration of nucleoli. Maintainance of their structural integrity as well as their de novo assembly mitosis following requires the activity of the rRNA genes. Nascent transcripts seem to key role in these processes. Since play a we could locate fibrillarin, a protein constituent of U3 RNP, to the free ends of growing transcripts we propose that the 5'regions of nascent pre-rRNA molecules link the active genes of the FCs to the surrounU. and ding nucleolar components (Scheer, Benavente, R., BioEssays 12, 14-21, 1990).
MOLECULAR MECHANISMS REGULATING RISOSOMAL GENE TRANSCRIPTION IN MAMMALIAN CELLS lngrid Grummt, Andraas Schnapp and Anne Kuhn. Institute of Biochemistry Rlrntgenring 11. 8700 WOnburg, F.R.G. Mouse RNA polymerase I transcription involves at least three auxiliary transcription initiation factors, termed TIF-IA. TIFIB and TIF-IC. Factor TIF-IB has been purified to molecular homogeneity. It is a 44 kd DNA binding protein which specifically interacts with he core region (from -39 to -1) of the rDNA promoter and confers promoter specificity to RNA polymerase I. Control of rRNA synthesis in response to the physiological state of the calls is brought about by TIF-IA, a regulatory factor whose amount or activity correlates with cell proliferation. This growth-dependent factor TIF-IA interacts with RNA polymerase I, thus converting it into a transcriptionally active holoenzyme, which is able to initiate specifically at the rDNA promoter in the presence of the other transcription factors, TIF-IB and TIF-IC. In addition, regulation of rRNA synthesis appears to involve a specific posttranslational modification of RNA polymerase I which is required for transcription initiation. Treatment of RNA polymerase I with alkaline phosphatase abolishes specific initiation at the rDNA promoter without significantly affecting the polymerizing activity of the enzyme at nonspecific templates. The data propose an efficient and versatile mechanism of rDNA transcription regulation which enables the cell to quickly adapt ribosome biosynthesis lo a variety of extracellular signals. Furthermore, we show that the 140 nt repetitive sequence elements located upstream from the gene promoter function as polymerase l-specific transcription enhancers. These repeats stimulate transcription of an adjacent gene promoter in cis and compete for transcription in trans. indicating that the enhancer elements bind at least one common factor which is required for promoter activity.
s39
Reports, Vol. 14, Abstracts Supplement
S38
EVOLUTION
OF THE
rDNA
MULTIGENE
1990
FAMILY
John M. Hancock & Gabriel A. Dover, Dept. of Genetics, University of
Cambridge, Downing Street, CambridgeCB23EH,U.K. The rDNA of eukaryotesis a mu&gene family in which a high level of similarity exists betwwn copiesof its basic repeat unit, primarily becauseof the homogeneizingconsequencesof unequalcrossingover. How are essential rDNA functions maintained during homogeneization? Three examples of “molecular ccevolution” reveal how this might be achieved. 1. Promoter-Polymerase
Coevolution
An interspecific incompatibility often exists between rDNA promoters and RNA polymerase I complexes. Recent evidences in humans, wheat and Drosophila suggest that this arises through coevolution between a newly homogenized promoter variant and a polymerase I cofactor, and between different cofactors. 2. Expansion
Segment
Coevolution
The expansion segmentsof large subunit rRNAs coevolve in many species by the acquisition of similar short, interspersed sequence motifs, probably generated by DNA slippage. The set of epansion segments in one species share motifs that are generally distinct from those in another species. 3. Compensatory Mutation and Compensatory Slippage When a point mutation occurs within a secondary structural stem of an rRNA molecule, it is often compensated by a second mutations which restores the base pairing in that position. Similarly, repetitive motifs are incorporated in
such a way that pre-existing stems are increased in length by a process of compensatory slippage. These processes are difficult to understand in the context of a multigene family but may be explained if the family can tolerate the spread of an uncompensatedmutation before compensation ties place. References:
Dover, G.A. (1982) “Molecular drive. (I cohesive mode of species evolution” Nature 299: 111-117: Dover. G.A. & Flavell. R.H. (1984) “Molecular coevolution: DNA divergence &d the maintenance .of fur&m” Ckll 38: 622-623: Hancock.J.M. & Dover G.A. (1988) “Molecular coevolurion among cryptically simple expansion segmenrsof eukaryoric 26Sl28S rRNAs” Mol. Biol. Evol. 5: 377.391; Hancock,J.M. et al. (1988) “Evolution of the secondary SWUC~WPS and compensntory mutations of the ribosomol RNAs of Drosophila melanogaster” slippage Mol. Biol. Evol. 5: 393-414:Hancock& Dover (1990) “Compensatory in the evolurion of ribosomol RNA genes” Science. sub&led.
COORDINATE CONTROL OF RJBOSOMAL PROTEIN SYNTHRS ISINYRAST Rudi J. Planta, Willem H. Mager, Thijs M. Doorenbosch and Leon S. Kraakman. Biochemisch Laboratorium, Vrije Universiteit, de Boelelaan 1083, Amstuxlam. The Netherlands.
s40
Ribosomal protein synthesis @p-synthesis) in yeast is a coordinate process resulting in the equimolar production of ahout 80 different ribosomal proteins. The rate of q-synthesis is precisely adjusted to the celhzlar growth rate. Regulation of rp-synthesis oeews primuily at the transcriptional level. Transcription activation of the majority of the ‘p-genes is mediated through common upstream activating sequences. socalled RPO-boxes, which usually are located at a distance of 200-500 tap from the start cadon. These RFSboxes represent hiding sites for a transaiptional activator, called TUP or RAP. The concenuation of TUF parallels the cellular growth rate and eviti exists that the reqmsc in expression of q-genes upon nutritional changes is medkted by this factor. Recent findings indicate that TUP/RAP also activates other gene families iuvokd in dhlar growth rate. In add&ion, this multifunctional pOthlhhdStOthC~-~-andtC-iUyeast. Some other rp-gum (e.g. those cnmding S33 and L45) do notcontainanRpG-hox.Theyappeartobeactivatcdhyanother multifunctional protein, called ABFl or SUP, which hinds to a specific squence element. This multifimctional protein also aetivatesolhcrlgMef,audhladditionhindstothemathlg type silencer ad ARSclrments.