A new vision of ocean biogeochemistry after a decade of the Joint Global Ocean Flux Study (JGOFS).
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Carbon↗
Biomedical subjects
T Platt
Publications and source records attributed to T Platt.
Escherichia coli rho factor induces release of yeast RNA polymerase II but not polymerase I or III.
Purified RNA polymerase II (pol II) from the yeast Saccharomyces cerevisiae pauses without releasing at many locations during in vitro transcription. Pausing can be induced by intrinsic DNA sequence as well as by specific DNA bound proteins such as the RNA pol I termination factor, Reb1p, or lac repressor. Addition of rho termination factor from E. coli induces RNA pol II to release at all of these pause sites. Rho-induced release of pol II requires both a rho binding site in the transcript upstream of the pause sites as well as hydrolysis of ATP. In contrast, rho factor has no effect on either pausing or release by RNA pol I or III. When combined with previous observations, these results suggest that RNA pol II may terminate by a mechanism closely related to the rho-dependent mechanism of prokaryotes. In contrast, pol I and III appear to utilize a mechanism more related to the rho-independent terminators of prokaryotes.
Adenosine Triphosphate↗
Do behavior changes herald physical illness in adults with mental retardation?
A longitudinal study of 62 individuals with profound mental retardation was conducted to determine if direct care staff can identify behavior change prior to identifying symptoms of acute illness. Results indicate that staff were able to notice changes in sluggishness prior to the onset of illness. Self-care behavior was of borderline significance and there was no significant change in eight behavior dimensions (vocalizations, peer conflict, stereotypy, aggression, self injurious behavior, restlessness, distractibility, and depression). This finding should alert physicians and caregivers to the importance of prompt response to symptoms. Reliance on behavioral observation of direct care staff is not always sensitive enough to pick up changes in health status in less restrictive residential environments.
Activities of Daily Living↗
Are individuals with mental retardation at high risk for chronic disease?
BACKGROUND AND OBJECTIVES: Family physicians are responsible for the health and illness care of individuals with mental retardation (MR) in many community practices. Therefore, it is important to determine the special disease patterns for this group. This study determined if individuals with MR are at increased risk for selected chronic diseases. METHODS: We analyzed 366 individuals, living in the community, with a primary diagnosis of MR. The two comparison groups without MR were 427 individual Medicaid recipients and 746 privately insured individuals. RESULTS: Individuals with MR had higher rates of neurophysical conditions (eg, seizures, central nervous system conditions, and sensory loss) compared to the other two groups. However, they had lower rates of some chronic conditions and health behaviors (eg, hypertension, migraines/chronic headaches, diabetes, depression or anxiety, obesity, substance abuse, and smoking) compared to other Medicaid and insured patients. CONCLUSIONS: Individuals with MR have lower rates of many chronic conditions, compared with other Medicaid recipients. This finding should reduce concerns among family physicians that treatment of this special population involves higher rates of chronic illness.
Chronic Disease↗
RNA binding analysis of yeast REF2 and its two-hybrid interaction with a new gene product, FIR1.
The product of the REF2 gene is required for optimal levels of endonucleolytic cleavage at the 3' ends of yeast mRNA, prior to the addition of a poly(A) tail. To test the role of the previously demonstrated nonspecific affinity of REF2 for RNA in this process, we have identified RNA binding mutants in vitro and tested them for function within the cell. One REF2 variant, with an internal deletion of 82 amino acids (269-350), displays a 10-fold reduction in RNA binding, yet still retains full levels of processing activity in vivo. Conversely, a series of carboxyl-terminal deletions that maintain full RNA binding capability have progressively decreasing activity. These results rule out a major role for the central RNA binding domain of REF2 in mRNA 3' end processing and demonstrate the importance of the carboxyl-terminal region. To ask if the stimulatory role of REF2 depends on interactions with other proteins, we used a two-hybrid screen to identify a new protein termed FIR1 (Factor Interacting with REF) encoded on chromosome V. FIR1 interacts with two independent regions of REF2, one of which (amino acids 268-345) overlaps the RNA binding domain and is dispensible for REF2 function, whereas the other (amino acids 391-533) is located within the critical carboxyl-terminus. As with REF2, FIR1 has a small but detectable role in influencing the efficiency of poly(A) site use. Yeast strains containing a disrupted FIR1 gene are slightly less efficient in the use of cryptic poly(A) sites located within the lacZ portion of an ACT1-lacZ reporter construct. Likewise, a double delta ref2, delta fir1 mutant is more defective in processing of a reporter CYC1 poly(A) site than delta ref2 alone. This synergistic response provides additional support for the interaction of FIR1 with REF2 in vivo, and suggests that a number of gene products may be involved in regulating the cleavage reaction in yeast.
Amino Acid Sequence↗
A mutation in the ATP binding domain of rho alters its RNA binding properties and uncouples ATP hydrolysis from helicase activity.
The Escherichia coli mutant rho201 was originally isolated in a genetic screen for defects in rho-dependent termination. Cloning and sequencing of this gene reveals a single phenylalanine to cysteine mutation at residue 232 in the ATP binding domain of the protein. This mutation significantly alters its RNA binding properties so that it binds trp t', RNA 100-fold weaker than the wild type protein, with a Kd of approximately 1.3 nM. Rho201 binds nonspecific RNA only 3-4-fold less tightly than it binds trp t', while the wild type differential for these same RNAs is 10-20-fold. Curiously, rho201 displays increased secondary site RNA activation, with a Km for ribo(C)10 of 0.6 microM, compared to the wild type value of 3-4 microM. Although rho201 and the wild type protein hydrolyze ATP similarly with poly(C), or trp t' RNA, as cofactors, rho201 has a higher ATPase activity when activated by nonspecific RNA. Physically, rho201 displays an abnormal conformation detectable by mild trypsin digestion. Despite effective ATP hydrolysis, the rho201 mutant is a poor RNA:DNA helicase and terminates inefficiently on trp t'. The single F232C mutation thus appears to uncouple the protein's ATPase activity from its helicase function, so rho can no longer harness available energy for use in subsequent reactions.
Adenosine Triphosphate↗
Analysis of E. coli rho factor: mutations affecting secondary-site interactions.
To define and differentiate primary and secondary RNA binding sites within the linear sequence of the rho protein, we investigated two mutant alleles, rho-115 and rhosuA1. They were first identified as defective in transcription termination in vivo, and later demonstrated to be defective in their interactions with RNA at the primary and secondary sites, respectively. Sequencing of rhosuA1 revealed a single lysine to glutamic acid residue change at position 352 (KE352), while rho-115 carries two mutations, glycine99 to valine (GV99) and a proline235 to histidine (PH235). Proteins carrying single mutations at each of these three positions were purified and their characteristics compared to the wild-type protein. We found both KE352 and GV99 to be defective in secondary-site RNA activation, with Km values for r(C)10 of 100 microM and approximately 650 microM, respectively, compared to the wild-type value of 4 microM. These observed secondary-site defects correlated with decreased helicase and ATPase activities, as well as a loss of transcription termination activity in vitro. By contrast, PH235 was very efficient at interacting with r(C)10 at the secondary site, with a measured Km of 0.5 microM, and displayed the characteristics of a hyperactive rho, as judged by its ATPase, helicase and termination capabilities. Our results show that mutations at three very different locations in the polypeptide can affect secondary-site activation by RNA, and that these interactions play a pivotal role in ATP hydrolysis, helicase activity and transcription termination.
Adenosine Triphosphatases↗
REF2 encodes an RNA-binding protein directly involved in yeast mRNA 3'-end formation.
The Saccharomyces cerevisiae mutant ref2-1 (REF = RNA end formation) was originally identified by a genetic strategy predicted to detect decreases in the use of a CYC1 poly(A) site interposed within the intron of an ACT1-HIS4 fusion reporter gene. Direct RNA analysis now proves this effect and also demonstrates the trans action of the REF2 gene product on cryptic poly(A) sites located within the coding region of a plasmid-borne ACT1-lacZ gene. Despite impaired growth of ref2 strains, possibly because of a general defect in the efficiency of mRNA 3'-end processing, the steady-state characteristics of a variety of normal cellular mRNAs remain unaffected. Sequencing of the complementing gene predicts the Ref2p product to be a novel, basic protein of 429 amino acids (M(r), 48,000) with a high-level lysine/serine content and some unusual features. Analysis in vitro, with a number of defined RNA substrates, confirms that efficient use of weak poly(A) sites requires Ref2p: endonucleolytic cleavage is carried out accurately but at significantly lower rates in extracts prepared from delta ref2 cells. The addition of purified, epitope-tagged Ref2p (Ref2pF) reestablishes wild-type levels of activity in these extracts, demonstrating direct involvement of this protein in the cleavage step of 3' mRNA processing. Together with the RNA-binding characteristics of Ref2pF in vitro, our results support an important contributing role for the REF2 locus in 3'-end processing. As the first gene genetically identified to participate in mRNA 3'-end maturation prior to the final polyadenylation step, REF2 provides an ideal starting point for identifying related genes in this event.
Amino Acid Sequence↗
A quaternary transcription termination complex. Reciprocal stabilization by Rho factor and NusG protein.
The Escherichia coli protein NusG is known to modulate Rho-dependent transcription termination in vivo. We have shown that it can also alter the pattern of Rho-dependent RNA endpoints in vitro, at lower NusG concentrations than can be explained by reported interactions between NusG and Rho or RNA polymerase. Three observations in vitro now suggest a model to account for these effects of NusG on Rho-dependent termination. First, the presence of NusG circumvents the interference with Rho function caused by adding DNA oligonucleotides complementary to particular segments of the Rho binding site. Second, when NusG is added to stalled elongation complexes, the off-rate of Rho from nascent RNA is slowed. Third, NusG associates stably with the elongation complex only when Rho is also present and bound to the nascent RNA. Our observations are consistent with a model in which NusG and Rho participate in an interdependent association with the transcribing RNA polymerase and the nascent RNA to facilitate the recognition and use of termination signals. Common structural and functional features shared with complexes that carry out processive antitermination are discussed.
Bacterial Proteins↗
Evidence supporting a tethered tracking model for helicase activity of Escherichia coli Rho factor.
Transcription termination factor Rho of Escherichia coli has an ATP-dependent RNA.DNA helicase activity that presumably facilitates RNA transcript release from the elongation complex. This helicase activity is unidirectional (5' to 3') and is stoichiometric, with one RNA molecule released per Rho hexamer in vitro. A simple RNA tracking model postulates that after Rho's initial binding, it translocates preferentially toward the 3' end of the RNA. Nitrocellulose filter binding studies combined with RNase H cleavage are inconsistent with this simple tracking model. Instead, they support a model in which Rho forms tight primary binding interactions with the recognition region of the RNA and remains bound there while transient secondary RNA binding interactions coupled to ATP hydrolysis serve to scan along the RNA to contact the RNA.DNA helix. This "tethered tracking" model is consistent with other properties of Rho factor, including the presence of two classes of RNA binding sites on the Rho hexamer and the 1:1 stoichiometry in the Rho helicase assay.
Adenosine Triphosphate↗
Poly(A) site selection in the yeast Ty retroelement requires an upstream region and sequence-specific titratable factor(s) in vitro.
In the Ty retrotransposon of Saccharomyces cerevisiae, as in most retroelements, the polyadenylation site of the 5' long terminal repeat (LTR) is ignored and the one in the 3' LTR is efficiently used. We examine here the contribution to this poly(A) site selection of the region termed 'U3', corresponding to the upstream non-transcribed portion of the 5' LTR. Using an established assay in vitro, we find that 3' processing is accurate and efficient with an RNA substrate corresponding to most of the LTR, whereas none is detectable with a shorter transcript lacking the U3 region, thus explaining why the 5' poly(A) site is ignored in genomic Ty mRNA. When HIS4 coding RNA, representing 'non-specific' sequence, replaces the U3 region, the Ty polyadenylation site is activated to 50% of the wild-type level. Within one specific region (TS1) in U3, 90-95 nt upstream of the poly(A) site, the change of UAGUAU to UCGCAU reduces processing efficiency by half, to the non-specific level provided by other sequences or by a deletion of the TS1 region. Another region (TS2) near the poly(A) site appears to be independently responsible for the remaining half of the processing activity. Alteration of both TS1 and TS2 eliminates processing entirely. In competition assays, excess unlabeled U3, but not its mutated counterparts, reduces the processing of radiolabeled Ty mRNA, suggesting the involvement of some sequence-specific titratable factor(s) in the whole cell extract for U3-specific activation.(ABSTRACT TRUNCATED AT 250 WORDS)
Binding, Competitive↗
Rho and RNA: models for recognition and response.
Escherichia coli Rho factor is required for termination of transcription at certain sites by RNA polymerase. Binding to unstructured cytosine-containing RNA target sites, subsequent RNA-dependent ATP hydrolysis, and an RNA-DNA helicase activity that presumably facilitates termination, are considered essential for Rho function. Yet the RNA recognition elements have remained elusive, the parameters relating RNA binding to ATPase activation have been obscure, and the mechanistic steps that integrate Rho's characteristics with its termination function in vitro and in vivo have been largely undefined. Recent work offers new insights into these interactions with results that are both surprising and satisfying in the context of Rho's emerging structure. These include the requirements for binding and ATPase activation by a variety of RNA substrates, dynamic analyses of Rho tracking, helicase and termination activity, and the participation of a new factor (NusG) that interacts with Rho. Models for Rho function are considered in the light of these recent revelations.
Bacterial Proteins↗
Deletion analysis of the Escherichia coli rho-dependent transcription terminator trp t'.
We have analyzed with deletion mutations the Escherichia coli rho-dependent transcription terminator trp t'. This site, 211 nucleotides in length, contains multiple sequence elements that are recognized by rho. Among these multiple elements are two that together appear to be important for full transcription termination efficiency in vitro and are similar to the rutA and rutB sequence elements in the lambda tR1 rho-dependent terminator previously described by Chen and Richardson (1987). The results suggest for the first time that common functionally important elements are shared among rho-dependent terminators. Other regions of trp t' also appear to contribute to its functional efficiency, consistent with the idea that rho-dependent terminators contain dispersed and redundant recognition segments.
Base Sequence↗
Transcriptional arrest of yeast RNA polymerase II by Escherichia coli rho protein in vitro.
A promoter-independent assay utilizing poly(dC)-tailed DNA templates has revealed that Saccharomyces cerevisiae whole-cell extracts can be proficient for transcription by the endogenous yeast RNA polymerase II as well as for correct 3'-end RNA processing. Our attempts to examine the fate of polymerase II itself were inconclusive, because only trace transcription products corresponded to the expected size of terminated RNA species. Transcription in our processing-proficient extract was thus insufficient to cause termination. To test our system with a known, albeit heterologous, signal, we examined a dC-tailed template carrying the E. coli rho-dependent termination signal trp t' in the yeast extract. Transcripts from this template were not susceptible to processing, but addition of rho protein resulted in two distinct truncated transcripts that could not be chased by excess unlabeled nucleotides. These RNA species thus represented stably paused or terminated polymerase II products, and their absence when a mutated unresponsive trp t' template was used affirmed that they were due to the effects of rho. E. coli RNA polymerase added to a yeast extract pretreated with alpha-amanitin was also halted by rho at these same two sites. A mutated rho protein, while only partly defective with E. coli polymerase, failed to provoke arrest when transcription was carried out by RNA polymerase II. Thus, functional rho and its cognate site, trp t', appear necessary and sufficient to elicit the production of truncated transcripts by RNA polymerase II in a yeast whole-cell extract. The ability of rho to halt the eukaryotic enzyme strengthens the likelihood that a rho-like helicase may be involved in RNA polymerase II transcription termination.
DNA-Directed RNA Polymerases↗
Selective enrichment of RNA species for tight binding to Escherichia coli rho factor.
We have applied the SELEX procedure (systematic evolution of ligands by exponential enrichment) to obtain RNA molecules that bind tightly to the Escherichia coli transcription termination factor rho. The starting pool was a population of RNA molecules 77 nucleotides (nt) long, in which was embedded a cassette of 30 nt of randomized sequence. The apparent dissociation constant of this RNA pool for hexameric rho factor was about 1 microM. After eight rounds of selection by filter binding, with RNA in either 10-fold or 40 to 100-fold excess at each step, the dissociation constant of the selected RNA had dropped by more than 500-fold to about 1 nM. Analysis of 29 clonal isolates from the population revealed that five had KDs substantially weaker than 10 nM (presumably background carryover), 40% were C-rich (as might have been predicted from rho's known substrate binding), and 40% had a strikingly preserved potential hairpin, in most cases of 6 base pairs with a 3 nt CAA loop and preceded by a CCCCA consensus. The rho-dependent trp t' terminator region includes a related potential hairpin structure; however, it is energetically unfavorable. The implications of the sequence findings for elucidating both static and dynamic aspects of rho factor recognition and response to its RNA target site are discussed.
Base Sequence↗
NusG alters rho-dependent termination of transcription in vitro independent of kinetic coupling.
To complement the recent discovery that rho-dependent termination in E. coli requires nusG protein in vivo, we have tested the effect of purified nusG protein on rho-dependent termination in vitro. With the well-characterized trp t' terminator of E. coli, and no other proteins than E. coli RNA polymerase and rho factor, nusG causes a proximal shift in the terminated RNA endpoints, compared to the endpoints generated by rho alone. The presence of nusG also enhances rho-mediated termination on partially defective mutant trp t' templates. We rule out explanations such as a change in the kinetic coupling between rho and RNA polymerase or a nusG-mediated increase in the affinity of rho for RNA. We also detect no difference in the helicase rate of rho in the presence of nusG. Even assays with completely stalled and isolated ternary complexes indicate that rho is able to effect the release of RNA with the assistance of nusG at points preceding the most proximal release sites observed in the absence of nusG. Our observations support a model in which nusG acts as a component of the transcription complex, possibly interacting with both rho and RNA polymerase as it governs accessibility to the nascent transcript.
Bacterial Proteins↗
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↗
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↗