Overproduced rho factor from p39AS has lysine replacing glutamic acid at residue 155 in the linker region between its RNA and ATP binding domains.
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Adenosine Triphosphate↗
Biomedical subjects
T Platt
Publications and source records attributed to T Platt.
Effects of decreased cytosine content on rho interaction with the rho-dependent terminator trp t' in Escherichia coli.
We have introduced multiple cytosine-to-uracil mutations in the rho-dependent transcription terminator trp t' and have characterized a subset of the resulting mutant derivatives in vitro for termination efficiency, affinity for rho, and stimulation of rho-ATPase activity. No specific cytosine residue appears to be required for termination, and at least 13 of the 28 cytosine residues in the 104 nucleotide trp t' region can be mutated with little effect on termination efficiency. One derivative with 11 mutations is significantly less efficient than other derivatives with a similar number of mutations, implying that the pattern of alterations, as well as the number, can affect termination efficiency. Derivatives with 20 and 28 cytosines mutated are non-functional in termination, consistent with the idea that cytosines are required for rho-dependent termination. Our results are inconsistent, however, with the hypothesis that the termination efficiency is directly related to the cytosine/guanine ratio in the nascent transcript. The results support the idea that neither RNA binding affinity nor ATPase activation per se are accurate predictors of rho-dependent termination efficiency.
Adenosine Triphosphatases↗
Unusual aspects of in vitro RNA processing in the 3' regions of the GAL1, GAL7, and GAL10 genes in Saccharomyces cerevisiae.
A striking feature of the 3'-end regions in polymerase II transcripts of Saccharomyces cerevisiae adjacent to their processing and polyadenylation sites is the lack of well-defined signal elements. Nonetheless, essential signals have seemed to be confined to compact regions in vivo, and we find that a short RNA with only 70 bases of GAL7 sequence upstream and 8 to 10 bases downstream of the poly(A) addition site is processed in vitro, as is an analogous CYC1 pre-RNA. Specific polyadenylation of a precleaved species further delimits the poly(A) signal and rules out obligatory coupling between cleavage and poly(A) addition. Although little proximal and even less distal sequence is required for accurate cleavage with CYC1 and GAL7, we have been unable to identify common features to which processing could be ascribed. We therefore turned to the coregulated set of genes in the galactose cluster (GAL1, GAL7, and GAL10) to assay their corresponding pre-mRNAs in vitro, in hopes of finding a common theme. By contrast to GAL7, short pre-mRNAs corresponding to GAL1 and GAL10 fail to be cleaved detectably, and only much longer transcripts are susceptible to processing. This indicates that signals, even if preserved, are more widely dispersed than the poly(A) addition site, and these results are unchanged whether extracts are from cells grown on glucose or galactose. As a further surprise, RNAs corresponding to the antisense orientation of the 3'-end regions of all three GAL genes are also effective substrates for the processing machinery in vitro. Computer analysis reveals the presence of polydisperse dyad symmetries that might account for these observations.
Base Sequence↗
Mutations in an RNP1 consensus sequence of Rho protein reduce RNA binding affinity but facilitate helicase turnover.
Escherichia coli rho protein facilitates transcription termination by a mechanism that involves rho binding to the nascent RNA, activation of rho's RNA-dependent ATPase activity, and release of the mRNA from the DNA template. The initial step, formation of a rho-RNA complex, is mediated primarily by an RNA binding domain included within the amino-terminal 151 amino acids of rho protein. We have now identified one specific portion of this region that is involved in RNA binding, by photocross-linking and by site-directed mutagenesis. UV irradiation of rho-RNA complexes results in covalent attachment of the RNA to a single peptide in rho that apparently spans amino acids 45-100. Within this peptide is a ribonucleoprotein (RNP1) consensus sequence, Gly-Phe-Gly-Phe, that is present in many RNA-binding proteins. Mutagenesis of the phenylalanine residues in this consensus to leucine or alanine results in mutant proteins that are defective for RNA binding and have altered ATPase and RNA-DNA helicase activities. The weakened affinity but increased salt sensitivity of RNA binding by the mutant proteins suggests that they have lost more than just a set of nonionic interactions and are consistent with a change in the conformation of the RNA binding site. Whatever the changes, they appear localized primarily to the RNA binding domain because the mutants retain much of their RNA-dependent ATPase activity. We infer that the Phe residues themselves do not play a substantial role in the activation of ATP hydrolysis. Our results indicate that several different components of RNA interaction are required for rho activity and support a role for the RNP1 consensus region of rho in at least one specific aspect of RNA binding.
Adenosine Triphosphatases↗
Polymerase chain reaction mapping of yeast GAL7 mRNA polyadenylation sites demonstrates that 3' end processing in vitro faithfully reproduces the 3' ends observed in vivo.
In general, synthetic RNA transcripts corresponding to the 3' ends of Saccharomyces cerevisiae genes appear to be accurately cleaved and polyadenylated in vitro under appropriate conditions in yeast cell extracts. Initially, however, the endpoints observed in vitro for the GAL7 gene failed to correlate adequately with those reported in vivo as derived from traditional S1 nuclease protection analyses. This led us to apply an independent method for analyzing mRNA 3' ends, using the polymerase chain reaction, with a first strand primer that incorporated a BamHI restriction site sequence near its 5' end, followed by (dT)17. This proved to be a sensitive and accurate means for determining precisely the major and minor polyadenylation sites of the GAL7 mRNA. Moreover, there was complete agreement between the sites identified with this technique when applied to cellular RNA and those generated in vitro by our 3' end mRNA processing reaction. This provides further support for the likelihood that processing in vitro faithfully reflects the endonucleolytic cleavage and polyadenylation events that occur within the living cell.
Base Sequence↗
Homologous mRNA 3' end formation in fission and budding yeast.
Sequences resembling polyadenylation signals of higher eukaryotes are present downstream of the Schizosaccharomyces pombe ura4+ and cdc10+ coding regions and function in HeLa cells. However, these and other mammalian polyadenylation signals are inactive in S. pombe. Instead, we find that polyadenylation signals of the CYC1 gene of budding yeast Saccharomyces cerevisiae function accurately and efficiently in fission yeast. Furthermore, a 38 bp deletion which renders this RNA processing signal non-functional in S. cerevisiae has the equivalent effect in S. pombe. We demonstrate that synthetic pre-mRNAs encoding polyadenylation sites of S. pombe genes are accurately cleaved and polyadenylated in whole cell extracts of S. cerevisiae. Finally, as is the case in S. cerevisiae, DNA sequences encoding regions proximal to the S. pombe mRNA 3' ends are found to be extremely AT rich; however, no general sequence motif can be found. We conclude that although fission yeast has many genetic features in common with higher eukaryotes, mRNA 3' end formation is significantly different and appears to be formed by an RNA processing mechanism homologous to that of budding yeast. Since fission and budding yeast are evolutionarily divergent, this lower eukaryotic mechanism of mRNA 3' end formation may be generally conserved.
Base Sequence↗
A short intervening structure can block rho factor helicase action at a distance.
We have characterized the helicase activity of transcription termination factor rho on a variety of substrates. Helicase activity requires specific recognition of a single-stranded region of RNA upstream (5') of the nucleic acid duplex on which rho acts. Spacer sequences of at least 450 nucleotides can be inserted between the rho-binding signals and the duplex region with little effect on activity. RNA-DNA helices of up to 120 base pairs, but not as long as 210 base pairs, can be disrupted efficiently by rho. The stoichiometry of release of substrates with long spacer sequences, as with the standard substrate, approaches a value of one RNA released per rho hexamer; thus cooperative binding by rho does not account for action at a distance. Instead, these results are consistent with a model in which a single rho hexamer binds initially to terminator sequences and then either loops out or tracks along the intervening RNA to reach the duplex region. Results with complex substrates are inconsistent with looping and support the tracking model: under conditions that allow disruption of RNA-DNA, but not RNA-RNA helices (0.4 mM Mg2+), the presence of a short RNA-RNA helix acts as a block to the disruption of an RNA-DNA helix downstream. These findings are discussed in relation to the mechanism of the helicase activity as well as its role in rho-dependent transcription termination.
Adenosine Triphosphatases↗
Specificity and efficiency of rho-factor helicase activity depends on magnesium concentration and energy coupling to NTP hydrolysis.
The RNA-DNA helicase activity of Escherichia coli transcription termination factor rho can be significantly enhanced at lower potassium chloride and magnesium acetate concentrations than previously used. Decreasing the potassium chloride concentration from 150 to 50 mM increases the rate of release at least 4-fold, while at lower magnesium concentrations less ATP is required for maximal duplex disruption. For all concentrations tested (between 0.1 and 5 mM), the optimal magnesium and ATP concentrations are interdependent; a roughly equimolar ratio gives the maximal rate of RNA release, although peak height and breadth vary. Surprisingly, rho behaves differently with an RNA-RNA duplex, which cannot be efficiently disrupted at magnesium concentrations below 1 mM. Above 2.0 mM, release does occur efficiently suggesting that Mg2+ promotes some structural transition in the RNA-RNA helix to a rho-susceptible conformation. In addition to Mg2+, helicase activity requires hydrolysis of nucleoside triphosphates, but for all four standard NTPs the rates of NTP hydrolysis do not correlate uniformly with the rates of RNA release. Based on the ratio of the rate of RNA release to the rate of NTP hydrolysis, rho utilizes ATP most efficiently. The 2-4-fold weaker coupling of hydrolysis to duplex disruption for the other three NTPs demonstrates that NTP utilization is not, on its own, sufficient for efficient helicase activity. The less efficient coupling with GTP, CTP, and UTP correlates with conformational differences in the protein complex as probed by mild trypsin digestion. The implications of our findings for substrate specificity and energy coupling in the helicase reaction are discussed.
Adenosine Triphosphatases↗
Sex determination and the Y chromosome: the application of molecular genetic technique to behavioral genetics.
In mammals, the Y chromosome mediates both gonadogenesis and spermatogenesis. It is also known to influence such traits as histocompatibility, sperm head morphology, pubertal (but not adult) testosterone level, sexual behavior, and aggressive behavior. An immediate goal in my laboratory is the isolation and characterization of the Y chromosomal gene responsible for initiating differentiation of the primitive bipotential gonads to become testes: the Y chromosomal gonadogenesis gene. Function of this gene initiates a cascade of events involving large numbers of other genes scattered throughout the genome, but it is not responsible for initiating development of all of the male phenotype; where : is XXSxr karyotype males, bearing the Sxr region of the Y chromosome which includes this gene, are sterile. It is not known if this gene influences those behaviors known to be influenced by the Y chromosome. If animals with an XXSxr karyotype, transgenic for specific Y chromosomal genes, could be produced, questions such as this could be answered. The developmental biology of the testis, molecular genetics of the Sxr region of the Y chromosome, and isolation of the testis determination gene from DNA of XXSxr males are discussed. Also discussed are the production of transgenic mice and the prospects for using such animals as coisogenic strains, differing by precisely known DNA sequences, in behavior genetic analysis. Such animals could be used both to test for behavioral phenotype and to dissect out biochemical and neurological mechanisms responsible for the behavior.
Animals↗
Mutations in the ATP-binding domain of Escherichia coli rho factor affect transcription termination in vivo.
Five mutant rho proteins, representing alterations at three different locations in the Escherichia coli rho gene that affect ATP hydrolytic activity but not RNA binding, were examined in vivo for function at the rho-dependent IS2 and bacteriophage lambda tR1 terminators. The altered amino acids in rho are located at highly conserved residues near the beta 1 and beta 4 strands of the hydrophobic ATP-binding pocket that is structurally similar to the F1-type ATPases and adenylate kinase. The RNA-dependent ATPase activities of the mutant rho proteins were previously shown to range from undetectable to a twofold increase over wild-type rho in vitro. Analysis of these proteins within the environment of the cell confirmed that transcription termination in vivo is indeed related to the ability of rho factor to properly hydrolyze nucleoside triphosphates, as would be predicted from results in vitro. The relative efficiency of termination at lambda tR1, as judged by lambda N= plating efficiency and by suppression of polarity of IS2 upstream of galK, was closely linked to the level of RNA-dependent ATPase activity observed in vitro for each protein. Moreover, the termination efficiency of four of the altered rho proteins at IS2 and lambda tR1 in vivo corresponded directly to the effect of these mutations on rho function at the E. coli trp t' terminator in vitro. We conclude that determinations of rho function in vitro accurately reflect its behavior in intracellular termination events.
Adenosine Triphosphate↗
RNA processing in vitro produces mature 3' ends of a variety of Saccharomyces cerevisiae mRNAs.
Ammonium sulfate fractionation of a Saccharomyces cerevisiae whole-cell extract yielded a preparation which carried out correct and efficient endonucleolytic cleavage and polyadenylation of yeast precursor mRNA substrates corresponding to a variety of yeast genes. These included CYC1 (iso-1-cytochrome c), HIS4 (histidine biosynthesis), GAL7 (galactose-1-phosphate uridyltransferase), H2B2 (histone H2B2), PRT2 (a protein of unknown function), and CBP1 (cytochrome b mRNA processing). The reaction processed these pre-mRNAs with varying efficiencies, with cleavage and polyadenylation exceeding 70% in some cases. In each case, the poly(A) tail corresponded to the addition of approximately 60 adenosine residues, which agrees with the usual length of poly(A) tails formed in vivo. Addition of cordycepin triphosphate or substitution of CTP for ATP in these reactions inhibited polyadenylation but not endonucleolytic cleavage and resulted in accumulation of the cleaved RNA product. Although this system readily generated yeast mRNA 3' ends, no processing occurred on a human alpha-globin pre-mRNA containing the highly conserved AAUAAA polyadenylation signal of higher eucaryotes. This sequence and adjacent signals used in mammalian systems are thus not sufficient to direct mRNA 3' end formation in yeast. Despite the lack of a highly conserved nucleotide sequence signal, the same purified fraction processed the 3' ends of a variety of unrelated yeast pre-mRNAs, suggesting that endonuclease cleavage and polyadenylation may produce the mature 3' ends of all mRNAs in S. cerevisiae.
Cytochrome b Group↗
Flow cytometry in oceanography.
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Flow Cytometry↗
Site-directed alterations in the ATP-binding domain of rho protein affect its activities as a termination factor.
We have utilized oligonucleotide site-directed mutagenesis to test our prediction that Escherichia coli rho factor has an ATP-binding domain separate from its RNA-binding domain and similar to that of adenylate kinase. Single amino acid substitutions were generated in regions thought to be within the active site and catalytically important for the ATPase activity, changing lysine 181 and/or lysine 184 to glutamine, and aspartate 265 to valine and asparagine. The altered proteins were purified and characterized in vitro for RNA- and ATP-binding ability, ATPase activity, helicase activity, and ability to catalyze transcription termination. Our results indicate that 1) these amino acid alterations in the proposed ATP-binding domain do not interfere with RNA binding; 2) substitution of lysine 184 by glutamine actually improves the ATPase and related activities while the same substitution at lysine 181 reduces but does not eliminate activity; 3) the double mutation changing both lysine 181 and lysine 184 to glutamine eliminates ATPase activity; and 4) the aspartate at 265 is also required for ATP hydrolysis but not for ATP binding. These results are consistent with our proposal that the general tertiary structure of rho's ATP-binding domain is similar to that of adenylate kinase.
Adenosine Triphosphatases↗
The ATP binding site on rho protein. Affinity labeling of Lys181 by pyridoxal 5'-diphospho-5'-adenosine.
We have labeled the nucleoside triphosphate-binding domain of Escherichia coli rho factor with the ATP affinity analog [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP). PLP-AMP completely inactivates the RNA-dependent ATPase activity of rho upon incorporation of 3 mol of reagent/mol of hexameric rho protein. Although the potency of PLP-AMP is enhanced when an RNA substrate such as poly(C) is present, the stoichiometry for inhibition remains the same as in the absence of poly(C). The nucleotide substrate ATP competes very effectively for the binding site and protects against PLP-AMP inactivation. A domain of rho called N2, which comprises the distal two-thirds of the molecule (residues 152-419) and encompasses the region proposed to bind ATP, is labeled specifically in the presence of poly(C). Amino acid sequence analysis of the single [3H]PLP-AMP labeled proteolytic fragment showed Lys181 to be the site of modification, suggesting that this residue normally interacts with the gamma-phosphoryl of bound ATP. These results agree with our proposed tertiary structure for the ATP-binding domain of rho that places this lysine residue in a flexible loop above a hydrophobic nucleotide-binding pocket comprised of several parallel beta-strands, similar to adenylate kinase, F1-ATPase, and related ATP-binding proteins. Parallel studies of rho structure and function by site-directed mutagenesis and chemical modification support this interpretation.
Adenosine Diphosphate↗
RNA processing generates the mature 3' end of yeast CYC1 messenger RNA in vitro.
In whole cell extracts of Saccharomyces cerevisiae, incubation of precursor mRNA transcripts encoding the sequences essential in vivo for forming the 3' end of the iso-1-cytochrome c mRNA (CYC1) revealed an endonuclease activity with the characteristics required for producing the mature mRNA 3' end. The observed cleavage in vitro is (i) accurate, occurring at or near the polyadenylation site of CYC1 RNA, (ii) 30 to 50 percent efficient, (iii) adenosine triphosphate dependent, (iv) specific for the 3' ends of at least two yeast pre-mRNA's, and (v) absent with related pre-mRNA's carrying mutations that abolish correct 3' end formation in vivo. In addition, a second activity in the extract polyadenylates the product under appropriate conditions. Thus, the mature 3' ends of yeast mRNA's may be generated by endonucleolytic cleavage and polyadenylation rather than by transcription termination.
Cytochrome c Group↗
Signals sufficient for rho-dependent transcription termination at trp t' span a region centered 60 base pairs upstream of the earliest 3' end point.
We have characterized the functional attributes of a 211-base pair region containing the Escherichia coli tryptophan operon rho-dependent terminator trp t', utilizing a series of constructs that alter the orientation, location, or extent of the trp t' sequences with respect to the trp promoter. In each instance, the extent of the rho-dependent response was monitored in vivo by read-through expression into a distal galactokinase gene and was compared with the results of transcription assays in vitro. As expected, transcription termination in vivo is dependent on the proper orientation of the terminator, and a tandem repeat of the terminator increases termination proportionally. Placing the terminator fragment only 14 nucleotides from the promoter does not affect termination significantly, supporting the belief that sequences outside of the 211-base pair fragment itself are dispensable. One construct, which lacks 116 base pairs, including the region encoding the normal RNA end points, still reduces galK activity in vivo and terminates transcription in vitro. Our results indicate that the rho response depends primarily on sequences in this 95-base pair segment, causing RNA polymerase to terminate transcription in a region 15-45 nucleotides further downstream.
Base Sequence↗
Orientation-dependent function of a short CYC1 DNA fragment in directing mRNA 3' end formation in yeast.
We have cloned an 82-base-pair region spanning the site of normal 3' end formation of Saccharomyces cerevisiae CYC1 mRNA into an integrative vector carrying the 5' end of the actin gene (including its intron) fused in frame to HIS4ABC sequences. This vector can confer the ability to grow on histidinol if HIS4C (encoding histidinol dehydrogenase) is sufficiently expressed. With the CYC1 fragment cloned in its wild-type (forward) orientation within the actin intron, transformants cannot grow on histidinol, whereas cells transformed with the vector carrying the reverse orientation of this fragment are able to grow well. RNA transfers demonstrate that transformants containing the forward orientation accumulate less than 40% of the control level of full-length mRNA and reveal the presence of a short, stable (approximately equal to 300 nucleotides) poly(A) RNA that represents 60-70% of the transcripts originating from the same promoter. The reverse orientation of the insert allows near-normal levels of full-length mRNA. Mapping of the 3' end of the truncated RNA indicates that poly(A) addition is variable in length but occurs at the same location as in the normal CYC1 transcript. Dominant and recessive suppressor mutations permit growth on histidinol despite the inserted fragment. Genetic analyses indicate that most of the dominant mutants are cis-acting and that the recessive mutants define a minimum of three complementation groups, indicating that defects in several different genes can restore higher levels of HIS4C expression.
Actins↗