Paradoxical aortic stiffening and subsequent cardiac dysfunction in Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is an ultra-rare disorder with devastating sequelae resulting in early death, presently thought to stem primarily from cardiovascular events. We analyse novel longitudinal cardiovascular data from a mouse model of HGPS (LmnaG609G/G609G) using allometric scaling, biomechanical phenotyping, and advanced computational modelling and show that late-stage diastolic dysfunction, with preserved systolic function, emerges with an increase in the pulse wave velocity and an associated loss of aortic function, independent of sex. Specifically, there is a dramatic late-stage loss of smooth muscle function and cells and an excessive accumulation of proteoglycans along the aorta, which result in a loss of biomechanical function (contractility and elastic energy storage) and a marked structural stiffening despite a distinctly low intrinsic material stiffness that is consistent with the lack of functional lamin A. Importantly, the vascular function appears to arise normally from the low-stress environment of development, only to succumb progressively to pressure-related effects of the lamin A mutation and become extreme in the peri-morbid period. Because the dramatic life-threatening aortic phenotype manifests during the last third of life there may be a therapeutic window in maturity that could alleviate concerns with therapies administered during early periods of arterial development.

Nam1p, a protein involved in RNA processing and translation, is coupled to transcription through an interaction with yeast mitochondrial RNA polymerase.

Alignment of three fungal mtRNA polymerases revealed conserved amino acid sequences in an amino-terminal region of the Saccharomyces cerevisiae enzyme implicated previously as harboring an important functional domain. Phenotypic analysis of deletion and point mutations, in conjunction with a yeast two-hybrid assay, revealed that Nam1p, a protein involved in RNA processing and translation in mitochondria, binds specifically to this domain. The significance of this interaction in vivo was demonstrated by the fact that the temperature-sensitive phenotype of a deletion mutation (rpo41Delta2), which impinges on this amino-terminal domain, is suppressed by overproducing Nam1p. In addition, mutations in the amino-terminal domain result specifically in decreased steady-state levels of mature mitochondrial CYTB and COXI transcripts, which is a primary defect observed in NAM1 null mutant yeast strains. Finally, one point mutation (R129D) did not abolish Nam1p binding, yet displayed an obvious COX1/CYTB transcript defect. This mutation exhibited the most severe mitochondrial phenotype, suggesting that mutations in the amino-terminal domain can perturb other critical interactions, in addition to Nam1p binding, that contribute to the observed phenotypes. These results implicate the amino-terminal domain of mtRNA polymerases in coupling additional factors and activities involved in mitochondrial gene expression directly to the transcription machinery.

Mutational analysis of the RNA component of Saccharomyces cerevisiae RNase MRP reveals distinct nuclear phenotypes.

The 340-nucleotide RNA component of Saccharomyces cerevisiae RNase MRP is encoded by the single-copy essential gene, NME1. To gain additional insight into the proposed structure and functions of this endoribonuclease, we have extensively mutagenized the NME1 gene and characterized yeast strains expressing mutated forms of the RNA using a gene shuffle technique. Strains expressing each of 26 independent mutations in the RNase MRP RNA gene were characterized for their ability to grow at various temperatures and on various carbon sources, stability of the RNase MRP RNA and processing of the 5.8S rRNA (a nuclear function of RNase MRP). 11 of the mutations resulted in a lethal phenotype, six displayed temperature-conditional lethality, and several preferred a non-fermentable carbon source for growth. In those mutants that exhibited altered growth phenotypes, the severity of the growth defect was directly proportional to the severity of the 5.8S rRNA processing defect in the nucleus. Together this analysis has defined essential regions of the RNase MRP RNA and provides evidence that is consistent with the proposed function of the RNase MRP enzyme.

Stability of the mitochondrial genome requires an amino-terminal domain of yeast mitochondrial RNA polymerase.

Mitochondrial RNA (mtRNA) polymerases are related to bacteriophage RNA polymerases, but contain a unique amino-terminal extension of unknown origin and function. In addition to harboring mitochondrial targeting information, we show here that the amino-terminal extension of yeast mtRNA polymerase is required for a mtDNA maintenance function that is separable from the known RNA polymerization activity of the enzyme. Deletion of 185 N-terminal amino acids from the enzyme results in a temperature-sensitive mitochondrial petite phenotype, characterized by increased instability and eventual loss of the mitochondrial genome. Mitochondrial transcription initiation in vivo is largely unaffected by this mutation and expression of just the amino-terminal portion of the protein in trans partially suppresses the mitochondrial defect, indicating that the amino-terminal extension of the enzyme harbors an independent functional domain that is required for mtDNA replication and/or stability. These results suggest that amino-terminal extensions present in mtRNA polymerases comprise functional domains that couple additional activities to the transcription process in mitochondria.

Mitochondrial DNA maintenance in vertebrates.

The discovery that mutations in mitochondrial DNA (mtDNA) can be pathogenic in humans has increased interest in understanding mtDNA maintenance. The functional state of mtDNA requires a great number of factors for gene expression, DNA replication, and DNA repair. These processes are ultimately controlled by the cell nucleus, because the requisite proteins are all encoded by nuclear genes and imported into the mitochondrion. DNA replication and transcription are linked in vertebrate mitochondria because RNA transcripts initiated at the light-strand promoter are the primers for mtDNA replication at the heavy-strand origin. Study of this transcription-primed DNA replication mechanism has led to isolation of key factors involved in mtDNA replication and transcription and to elucidation of unique nucleic acid structures formed at this origin. Because features of a transcription-primed mechanism appear to be conserved in vertebrates, a general model for initiation of vertebrate heavy-strand DNA synthesis is proposed. In many organisms, mtDNA maintenance requires not only faithful mtDNA replication, but also mtDNA repair and recombination. The extent to which these latter two processes are involved in mtDNA maintenance in vertebrates is also appraised.

Autoinducer-independent mutants of the LuxR transcriptional activator exhibit differential effects on the two lux promoters of Vibrio fischeri.

The LuxR protein is a transcriptional activator which, together with a diffusible small molecule termed the autoinducer [N-(3-oxohexanoyl)-L-homo-serine lactone], represents the primary level of regulation of the bioluminescence genes in Vibrio fischeri. LuxR, in the presence of autoinducer, activates transcription of the luxICDABEG gene cluster and both positively and negatively autoregulates transcription of the divergently oriented luxR gene, activating transcription at low levels of autoinducer, and repressing synthesis at high autoinducer concentration. Seven LuxR point mutants which activate V. fischeri lux transcription in the absence of autoinducer (LuxR*) have been characterized. The LuxR* proteins activated transcription of the bioluminescence genes to levels 1.5-40 times that achieved by wild-type LuxR without autoinducer. All of the LuxR* mutants retained responsiveness to autoinducer. However, in each case the degree of stimulation in response to autoinducer was lower than that observed for wild-type LuxR. The LuxR* proteins retained the requirement for autoinducer for autoregulation of the luxR gene. We propose that the LuxR protein exists in two conformations, an inactive form, and an active form which predominates in the presence of autoinducer. The LuxR* mutations appear to shift the equilibrium distribution of these two forms so as to increase the amount of the active form in the absence of autoinducer, while autoinducer can still convert inactive to active species. The differential effects of the LuxR* proteins at the two lux promoters suggest that LuxR stimulates PluxR transcription by a different mechanism to that used at the PluxI promoter, implying that binding of LuxR to its binding site, known to be necessary for transcriptional activation, may not be sufficient.

Functional conservation of yeast mtTFB despite extensive sequence divergence.

Transcription of mtDNA in the yeast S. cerevisiae depends on recognition of a consensus nonanucleotide promoter sequence by mtRNA polymerase acting with a 40-kDa dissociable factor known as mtTFB or Mtflp. mtTFB has been cloned and characterized in S. cerevisiae, but has not been studied in similar detail in any other organism. Although it is known that mitochondrial transcription in the dairy yeast, Kluyveromyces lactis, initiates within the same consensus promoter sequence used in S. cerevisiae, no previous studies have focused on the proteins involved in transcription initiation in K. lactis. In this article, we report the cloning of mtTFB from K. lactis and from a yeast more closely related to S. cerevisiae, S. kluyveri. Both novel mtTFB genes were able to substitute for the MTF1 gene in S. cerevisiae. Both proteins purified following expression in E. coli were able to support specific transcription initiation in vitro with the S. cerevisiae mtRNA polymerase. The S. kluyveri and K. lactis mtTFB proteins share only 56% and 40% identity with S. cerevisiae mtTFB, respectively. Alignments of the three mtTFB sequences did not reveal any regions larger than 30 amino acids with greater than 60% amino acid identity. In particular, regions proposed to show sequence similarity to bacterial sigma factors were not more highly conserved than other regions of the mtTFB proteins. All three yeast mtTFB genes lack conventional amino-terminal mitochondrial targeting sequences, suggesting that all three proteins may be imported into mitochondria by the same unusual mechanism reported for S. cerevisiae mtTFB.

Addition of a 29 residue carboxyl-terminal tail converts a simple HMG box-containing protein into a transcriptional activator.

Human mitochondrial transcription factor A (h-mtTFA) is essential for initiation of transcription from the two promoters located in the displacement-loop region of human mitochondrial DNA. This 25 kDa protein contains two tandem, HMG box DNA-binding domains separated by a 27 amino acid residue linker region and followed by a 25 residue carboxyl-terminal tail; both the linker and tail are rich in basic amino acid residues. Mutational analysis of h-mtTFA revealed that the tail region is important for specific DNA recognition and essential for transcriptional activation. The critical role of the human tail in transcription was confirmed by constructing chimeric proteins that exchanged similar regions between h-mtTFA and its Saccharomyces cerevisiae homolog, sc-mtTFA. Wild-type sc-mtTFA is unable to activate transcription from the human mitochondrial light-strand promoter (LSP). Addition of the human tail region to sc-mtTFA conferred LSP-specific promoter activation. In all of the different h-mtTFA mutations tested, transcriptional activation was correlated with specific DNA-binding activity, suggesting that these two functions may be inseparable, a situation entirely consistent with previous mutational analyses of human mitochondrial promoters.

Human mitochondrial transcription factor A and promoter spacing integrity are required for transcription initiation.

The two major promoters for transcription of the human mitochondrial genome are located near each other in the displacement-loop region of the molecule. Previous work has localized these promoters to regions of < 100 nucleotides each; the DNA sequence at the transcription start site is stringently required, as is the region from -10 to -40 base pairs upstream of each respective start site. Each upstream site is recognized and bound by human mitochondrial transcription factor A (h-mtTFA), an event previously shown to be important for transcriptional activation. We report here results using recombinant h-mtTFA that demonstrate the dependence of transcription initiation of h-mtTFA. In addition, altering the distance between the h-mtTFA binding site and the transcription start site greatly impairs transcription initiation efficiency. The decrease in transcription initiation efficiency was shown to be a consequence of altering the position of h-mtTFA binding as opposed to the strength of h-mtTFA binding, as judged by DNA footprinting ability. Analysis of a chimeric yeast-human promoter revealed that the yeast mtTFA homologue cannot substitute for the human protein, even when bound at an appropriate position upstream of the human transcription start site.

A Saccharomyces cerevisiae mitochondrial transcription factor, sc-mtTFB, shares features with sigma factors but is functionally distinct.

In Saccharomyces cerevisiae mitochondria, sc-mtTFB is a 341-amino-acid transcription factor required for initiation of transcription from mitochondrial DNA promoters. Specific transcription in vitro requires only sc-mtTFB and the bacteriophage-related core sc-mtRNA polymerase. Mutational analysis of sc-mtTFB has defined two regions of the protein that are important for normal function both in vivo and in vitro. These regions overlap portions of the protein that exhibit similarity to conserved region 2 of bacterial sigma factors. One mutation in this region of sc-mtTFB (tyrosine 108 to arginine [Y108R]) has a defective phenotype that matches that observed for mutations in the corresponding residue of Bacillus subtilis sigma A and sigma E proteins. However, mutations in the sigma 2.4-like region, including a 5-amino-acid deletion corresponding to crucial promoter-contacting amino acids of sigma factors, did not eliminate the ability of sc-mtTFB to initiate transcription specifically in vitro. This suggests a mechanism of promoter recognition for sc-mtRNA polymerase different from that used by bacterial RNA polymerases. Two mutations in a basic region of sc-mtTFB resulted in defective proteins that were virtually dependent on supercoiled DNA templates in vitro. These mutations may have disrupted a DNA-unwinding function of sc-mtTFB that is only manifested in vitro and is partially rescued by DNA supercoiling.

Identification of a distantly located regulatory element in the luxD gene required for negative autoregulation of the Vibrio fischeri luxR gene.

Expression of bioluminescence in the marine bacterium Vibrio fischeri is controlled by a unique cell density-dependent regulatory mechanism called auto-induction. The genes required for bioluminescence (the lux genes) are organized in two divergently transcribed operons (luxR-luxICDABEG). One operon (luxICDABEG) contains the genes required for light production (luxCDABE) and the synthesis of a diffusible signal molecule called autoinducer (luxI). The other operon contains the luxR gene which encodes a transcriptional regulatory protein that activates transcription of both lux operons in the presence of autoinducer. This bidirectional stimulatory mechanism leads to a positive feedback circuit that results in a rapid increase in light production at a particular culture cell density which is characteristic of autoinduction. Transcriptional positive feedback is apparently limited by a negative autoregulatory circuit through which LuxR acts to inhibit its own synthesis. Transcriptional negative autoregulation requires autoinducer, the lux operator located in the control region (which is the binding site for LuxR), and negative acting DNA sequences in the luxICDABEG operon. Deletion analysis of the luxICDABEG operon demonstrated that a negative acting element is located in the luxD gene at a position 2.0 kilobases from the lux operator. The nucleotide sequence of this luxD element is similar to the lux operator (11 of 20 base pairs identical) and can function as a LuxR-binding site when it replaces the lux operator in the control region. These results suggest that the luxD element functions as a low affinity binding site for LuxR and that occupancy of this site is required to achieve transcriptional negative autoregulation of luxR.

Positive autoregulation of the Vibrio fischeri luxR gene. LuxR and autoinducer activate cAMP-catabolite gene activator protein complex-independent and -dependent luxR transcription.

The LuxR protein is a transcriptional activator involved in regulation of the genes required for bioluminescence (lux) in the marine bacterium Vibrio fischeri. Transcription of the two divergently oriented lux operons (luxR and luxICDABEG) is activated by LuxR in the presence of a diffusible inducer (autoinducer). Transcription of the luxR gene is subject to both positive and negative autoregulation as well as activation by the cAMP-catabolite gene activator protein complex (cAMP-CAP). Transcription of luxR was studied using both luminescence in vivo as a reporter and primer extension analysis of mRNA synthesized in vivo. Mutation of the lux CAP-binding site resulted in a reduction in luminescence from the reporter and the complete loss of luxR positive autoregulation. Positive autoregulation was restored if luxR was provided in trans, demonstrating that LuxR and autoinducer activate luxR transcription in the absence of cAMP-CAP. By means of primer extension analysis, three sites of initiation of luxR transcription were demonstrated; initiation at two of these sites required cAMP-CAP. The quantity of all three transcripts was increased in the presence of LuxR and autoinducer when a plasmid with a wild-type CAP-binding site was used. Initiation at the cAMP-CAP-dependent sites was not observed from a plasmid with a mutated CAP-binding site in the presence or absence of autoinducer even with luxR supplied in trans. Instead, with luxR supplied in trans, initiation at the cAMP-CAP-independent initiation site was specifically stimulated by LuxR and autoinducer. Thus, in the course of positive autoregulation, the LuxR protein activates transcription from two luxR promoters by a cAMP-CAP-dependent mechanism and a third promoter by a cAMP-CAP-independent mechanism.

The Vibrio fischeri LuxR protein is capable of bidirectional stimulation of transcription and both positive and negative regulation of the luxR gene.

Regulation of the genes required for bioluminescence in the marine bacterium Vibrio fischeri (the lux regulon) is a complex process requiring coordination of several systems. The primary level of regulation is mediated by a positive regulatory protein, LuxR, and a small diffusible molecule, N-(3-oxo-hexanoyl)-homoserine lactone, termed autoinducer. Transcription of the luxR gene, which encodes the regulatory protein, is positively regulated by the cyclic AMP-CAP system. The lux regulon of V. fischeri consists of two divergently transcribed operons designated operonL and operonR. Transcription of the rightward operon (operonR; luxICDABE), consisting of the genes required for autoinducer synthesis (luxI) and light production (luxCDABE), is activated by LuxR in an autoinducer-dependent fashion. The leftward operon (operonL) consists of a single known gene, luxR. The LuxR protein has also been shown to decrease transcription of operonL through an autoinducer-dependent mechanism, thereby negatively regulating its own synthesis. In this paper we demonstrate that the autoinducer-dependent repression of operonL transcription requires not only LuxR but also DNA sequences within operonR which occur upstream of the promoter for operonL. In the absence of these DNA sequences, the LuxR protein causes an autoinducer-dependent activation of transcription of operonL. The lux operator, located in the control region between the two operons, was required for both the positive and negative autoinducer-dependent responses. By titration of high levels of LuxR supplied in trans with synthetic autoinducer, we found that low levels of autoinducer could elicit a positive response even in the presence of the negative-acting DNA sequences, while higher levels of autoinducer resulted in a negative response. Without these DNA sequences in operonR, LuxR and autoinducer stimulated transcription regardless of the level of autoinducer. These results suggest that a switch between stimulation and repression of operonL transcription is mediated by the levels of the LuxR-autoinducer complex, which in these experiments reflects the level of autoinducer in the growth medium.

Use of regulated cell lysis in a lethal genetic selection in Escherichia coli: identification of the autoinducer-binding region of the LuxR protein from Vibrio fischeri ATCC 7744.

A lethal genetic selection utilizing the bacteriophage lambda lysis genes (S, R, RZ) has been developed and used in conjunction with a luminescence screen to allow the isolation and characterization of six missense mutations and two nonsense mutations in the luxR gene from Vibrio fischeri ATCC 7744. A transcriptional fusion of the lysis genes in operonR downstream of a truncated luxI gene allows control of cell lysis by the addition of synthetic autoinducer to the growth medium. The six missense mutations isolated resulted in changes in the LuxR protein of Asp at position 79 to Asn (hereafter designated as D79N), V82I, V109L, L118F, S123I, and H217Y. Variant LuxR proteins with amino acid changes of D79N, V82I, V82L, and H127Y were shown to require higher concentrations of autoinducer to elicit a certain amplitude response than is required by the wild-type protein. We believe that the clustering of a total of seven randomly generated missense mutations in a 49-amino-acid region of the LuxR primary sequence defines a critical portion of the LuxR protein. The observation that proteins with lesions in this region responded to elevated levels of autoinducer suggests that the autoinducer-binding site is constructed, at least in part, from several amino acid residues within the 79-to-127 region of the LuxR protein.