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PubMed2002

Regulation of the bacillus subtilis ccpC gene by ccpA and ccpC.

Bacillus subtilis CcpC, a LysR-type transcriptional regulator, represses the transcription of genes for citrate synthase (citZ) and aconitase (citB) in response to citrate availability. Transcription of ccpC was shown to initiate at two promoters, P1, located just upstream of the ccpC gene, and P2, located within or upstream of the neighbouring ykuL gene. Expression from the ccpC-specific promoter (P1) was negatively regulated by CcpC but independent of the carbon source in the medium. Gel shift and DNase I footprinting experiments revealed that CcpC binds to an interrupted dyad sequence that surrounds the ccpC transcriptional start point. Transcription of ccpC from the upstream promoter (P2) was repressed by glucose in a CcpA-dependent manner. A putative CcpA binding site (cre) was identified upstream of the -35 region of the P1 promoter. Transcriptional fusion studies demonstrated that glucose repression of ccpC expression from the P2 promoter depends on this cre site. In addition, DNase I footprinting experiments showed that CcpA specifically binds to this cre site and that the introduction of mutations (cre*) into this site abolished the binding. These results suggest that CcpA may control CcpC synthesis by acting as a road-block to readthrough transcription from the P2 promoter.

Bacillus subtilis↗
PubMed1995

A zinc-binding region in the beta' subunit of RNA polymerase is involved in antitermination of early transcription of phage HK022.

Antitermination of early transcription in phage HK022 requires no virus-encoded proteins and thus differs from antitermination by other lambdoid phages. It does require cis-acting phage sequences, which may be analogous to the lambdoid nut sites. To identify host proteins involved in antitermination, we isolated 14 Escherichia coli mutants that are specifically blocked in HK022 growth. The mutations are located in the rpoC gene, which encodes the beta' subunit of RNA polymerase. Each mutation alters one of three amino acid residues located within a cluster of four completely conserved cysteine residues that are believed to bind zinc. We examined the effect of one mutation on HK022 antitermination in vivo. rpoCY75N greatly reduced readthrough of a strong rho-independent transcription terminator placed downstream of the HK022 PL promoter and nutL analog, but did not decrease promoter activity. Purified enzyme had a similar effect on PL-directed transcription in vitro: wild-type but not mutant polymerase read through a strong rho-independent terminator located immediately downstream of the nutL analog with high efficiency. We suggest that interaction of the putative zinc-binding domain of the RNA polymerase beta' subunit with the HK022 antitermination sites suppresses transcription termination, and that this interaction can occur in the absence of other proteins.

Amino Acid Sequence↗
PubMed2002

Clinically relevant aminoglycosides can suppress disease-associated premature stop mutations in the IDUA and P53 cDNAs in a mammalian translation system.

Recent studies have suggested that the use of aminoglycosides to suppress disease-causing nonsense mutations may be a promising new therapy for a large number of genetic diseases. However, gentamicin is currently the only clinically relevant aminoglycoside shown to suppress premature stop mutations in a mammalian system. We compared the ability of the clinically approved aminoglycosides gentamicin, tobramycin, and amikacin to suppress premature stop mutations. Using readthrough reporter constructs as well as mammalian cDNAs containing naturally occurring premature stop mutations, we found that each of these aminoglycosides can suppress many premature stop mutations in a context-dependent manner in a mammalian translation system. Our results indicate that the tetranucleotide termination signal (the stop codon and the nucleotide 3' of the stop codon) is the primary determinant for aminoglycoside-mediated suppression. The levels of termination suppression achieved by tobramycin were substantially lower than those observed with gentamicin. In contrast, amikacin stimulated suppression in a manner that was generally similar to gentamicin. Amikacin produced higher levels of readthrough than gentamicin at some contexts, demonstrating a unique pattern of context dependence. Experiments with mammalian cDNAs confirmed these results and demonstrated that these aminoglycosides can also suppress disease-associated premature stop mutations previously identified in the IDUA gene (responsible for the lysosomal storage disease mucopolysaccharidosis I) and the P53 gene (associated with many forms of cancer). Taken together, these results suggest that amikacin represents an alternative to gentamicin for suppression therapy in certain contexts, thus providing a means of optimizing the efficacy of aminoglycoside-mediated suppression of premature stop mutations.

Amikacin↗
PubMed1987

Chromogenic identification of promoters in Streptomyces lividans by using an ampC beta-lactamase promoter-probe vector.

A promoter-probe system, based on the ampC beta-lactamase gene of Escherichia coli, has been developed for the isolation and characterization of transcriptional signals in the gram-positive bacterium Streptomyces lividans. The promoter-probe vector, denoted pJAS14, has the SLP1.2 replicon with a copy number of four-five plasmids per cell. It contains a unique BamHI site just in front of the ampC ribosome-binding site, and upstream of this BamHI site a transcriptional terminator signal that prevents readthrough transcription from plasmid-borne promoters has been inserted. Using pJAS14, we have shot-gun cloned chromosomal DNA from S. lividans and S. lavendulae into the BamHI site, and isolated a number of promoter containing DNA fragments by the use of the chromogenic cephalosporin nitrocefin. On plates, we identified promoters of varying strengths and also with differences in nutritional and temporal expression. Using liquid cultures of S. lividans, it has been demonstrated that one promoter, denoted P1 (SEP8), as well as the ampC gene of E. coli, show activity corresponding to the vegetative growth of the cells. The P1 (SEP8) promoter was shown to be expressed also in E. coli, and it initiates RNA synthesis at exactly the same nucleotides in both S. lividans and E. coli. The promoter shows good homology to the E. coli promoter consensus sequence in both the -35 and -10 regions. Thus, this promoter is a representative of the SEP (Streptomyces E. coli-type promoter) class of promoters recently described (Jaurin and Cohen 1985). This indicates that an S. lividans RNA polymerase recognizes the same sequence determinants and chooses the point of initiation of RNA synthesis in the same way as the corresponding E. coli enzyme.

Base Sequence↗
PubMed1996

The genes of lepA and hemN form a bicistronic operon in Bacillus subtilis.

The IepA operon of Bacillus subtilis was found to be bicistronic and to consist of the two genes IepA and hemN, which encode a putative GTP-binding protein and an oxygen-independent coproporhyrinogen III oxidase, respectively. The IepA operon is located immediately upstream of the dnaK operon. Both operons are transcribed in the same direction and are not separated by an obvious transcription-terminator-like structure. The IepA operon is preceded by a potential vegetative promoter, and there is a putative strong intergenic terminator between IepA and hemN. Northern blot experiments revealed only a transcript corresponding to IepA, but expression of hemN was demonstrated in slot-blot and immunoblot experiments using antibodies raised against His-tagged HemN. The data suggest that most of the transcripts originating at the potential vegetative promoter are terminated at the intergenic terminator. Readthrough transcription into the downstream dnaK operon was not found.

Amino Acid Sequence↗
PubMed1989

Termination of transcription of ribosomal RNA genes of mung bean occurs within a 175 bp repetitive element of the spacer region.

In mung bean (Vigna radiata, formerly Phaseolus aureus) one length heterogeneity in the intergenic spacer (IGS) of the ribosomal DNA (rDNA) is due to a variable number of 175-bp subrepeats. This spacer region downstream of the 25S rRNA coding region was characterized by sequencing the 2.4 kb EcoRI/HindIII fragment of a 10.5 kb mung bean rDNA repeat. Within the 175-bp repetitive elements a sequence was detected showing strong similarity to the T2/T3-box (GACTTGC) found in Xenopus rDNA and involved in termination and enhancing transcription. In mung bean this sequence partly forms the stem of a possible stem-loop structure at the 3'end of each subrepeat. Nuclease mapping of transcription termination sites (TTS) results in two signals, 65 bp and 315 bp downstream of the 3'end of the 25S rRNA coding region. The longer transcript terminates 20 bp downstream of the stem-loop structure at the end of the first 175-bp subrepeat. A spacer model is proposed which allows "readthrough enhancement". No cross-hybridization was observed between the 180-bp subrepeats in pea (Pisum sativum) rDNA and the mung bean 175-bp subrepeat.

Base Sequence↗
PubMed1995

A complete plasmid-based complementation system for RNA coliphage Q beta: three proteins of bacteriophages Q beta (group III) and SP (group IV) can be interchanged.

Our laboratory has established a bacteriophage Q beta cDNA-containing plasmid system in which virtually all coding defects present within the 4217 nucleotide Q beta genome can be complemented in trans. In this system, Q beta minus strand RNAs are constitutively transcribed from plasmid cDNA by Escherichia coli RNA polymerase. Replication of these minus strands results in the synthesis of Q beta plus RNA, thereby triggering an infectious cycle in which Q beta phase particles are generated. Genetically engineered Q beta genome mutations that result in defective viral proteins can be complemented in trans by the products of one or more Q beta helper plasmids that express either: (1) Q beta maturation protein, which can complement defects in the Q beta maturation cistron (nucleotides 61 to 1320); (2) Q beta readthrough protein, which can complement defects in the readthrough cistron (nucleotides 1344 to 2330); or (3) Q beta replicase, which can complement defects in the replicase cistron (nucleotides 2352 to 4118). Each plasmid component of this system contains a unique origin of replication and carries a different antibiotic gene, thereby enabling all combinations of these plasmids to coexist in the same host. We have further developed a second series of helper plasmids that generate the corresponding viral proteins of the related group IV RNA phage SP. Each of these SP helper proteins can complement respective defects within the Q beta genome with efficiencies similar to those observed for the Q beta helper proteins. It is now possible to supply functional Q beta or SP proteins in trans to examine Q beta genomes that contain protein coding defects for their ability to synthesize Q beta proteins, replicate Q beta RNA, assemble virions, and/or lyse the host cell.

Allolevivirus↗
PubMed1996

Identification of a novel selD homolog from eukaryotes, bacteria, and archaea: is there an autoregulatory mechanism in selenocysteine metabolism?

Escherichia coli selenophosphate synthetase (SPS, the selD gene product) catalyzes the production of monoselenophosphate, the selenium donor compound required for synthesis of selenocysteine (Sec) and seleno-tRNAs. We report the molecular cloning of human and mouse homologs of the selD gene, designated Sps2, which contains an in-frame TGA codon at a site corresponding to the enzyme's putative active site. These sequences allow the identification of selD gene homologs in the genomes of the bacterium Haemophilus influenzae and the archaeon Methanococcus jannaschii, which had been previously misinterpreted due to their in-frame TGA codon. Sps2 mRNA levels are elevated in organs previously implicated in the synthesis of selenoproteins and in active sites of blood cell development. In addition, we show that Sps2 mRNA is up-regulated upon activation of T lymphocytes and have mapped the Sps2 gene to mouse chromosome 7. Using the mouse gene isolated from the hematopoietic cell line FDCPmixA4, we devised a construct for protein expression that results in the insertion of a FLAG tag sequence at the N terminus of the SPS2 protein. This strategy allowed us to document the readthrough of the in-frame TGA codon and the incorporation of 75Se into SPS2. These results suggest the existence of an autoregulatory mechanism involving the incorporation of Sec into SPS2 that might be relevant to blood cell biology. This mechanism is likely to have been present in ancient life forms and conserved in a variety of living organisms from all domains of life.

Amino Acid Sequence↗
PubMed1997

Studies on the translation mechanism of subgenomic RNA of potato leafroll virus.

The expression of open reading frames located on the subgenomic RNA (sgRNA) has been studied in an in vitro transcription and translation system. The obtained results indicate: a) translation of sgRNA occurs according to the scanning model since the insertion of palindrome (delta G0 = -61 kcal/mol) prevents the initiation of translation; b) ORF6 is translated by suppression of the stop codon separating ORF4 from ORF6 and the presence of suppressor tRNA is necessary for the readthrough; c) the presence of leader sequence of sgRNA (212 nucleotides) decreases the translation efficiency of ORFs located downstream and it affects the ratio of products of ORF4 and ORF5; d) 3'UTR does not influence on an expression of genes located on the sgRNA.

Base Sequence↗
PubMed2004

Effects of altering the transcription termination signals of respiratory syncytial virus on viral gene expression and growth in vitro and in vivo.

Nonsegmented negative-sense RNA viruses (mononegaviruses) control viral gene expression largely through a transcription gradient such that promoter-proximal genes are transcribed more abundantly than downstream genes. For some paramyxoviruses, naturally occurring differences in the levels of efficiency of transcription termination by various gene end (GE) signals provide an additional level of regulation of gene expression. The first two genes (NS1 and NS2) of respiratory syncytial virus (RSV) are particularly inefficient in termination. We investigated whether altering the termination efficiency (TE) of these two genes in infectious recombinant virus would affect transcription of promoter-proximal and promoter-distal genes, production of viral proteins, and viral replication in cell culture and in the respiratory tract of mice. Recombinant RSVs were constructed with mutations that increased or decreased the TE of the NS1 GE signal, increased that of the NS2 GE signal, or increased that of both signals. Increasing the TE of either or both GE signals resulted in decreased production of the related polycistronic readthrough mRNAs, which normally arise due to the failure of the viral polymerase to recognize the GE signal. This was accompanied by a small increase in the levels of monocistronic NS1 and NS2 mRNAs. Conversely, decreasing the TE of the NS1 GE increased the production of readthrough mRNAs concomitant with a decrease of monocistronic NS1 and NS2 mRNA levels. These changes were reflected in the levels of NS1 and NS2 protein. All of the mutant viruses displayed growth kinetics and virus yields similar to wild-type recombinant RSV (rA2) in both HEp-2 and Vero cells. In addition, all mutants grew similarly to rA2 in the upper- and lower-respiratory tract of BALB/c mice, though some of the mutants displayed slightly decreased replication. These data suggest that the natural inefficiencies of transcription termination by the NS1 and NS2 GE signals do not play important roles in controlling the magnitude of RSV gene expression or the efficiency of virus replication. Furthermore, while changes in the TE of a GE signal clearly can affect the transcription of its gene as well as that of the one immediately downstream, these changes did not have a significant effect on the overall transcriptional gradient.

Animals↗
PubMed1992

Photosystem II function and integrity in spite of drastic protein changes in a conserved region of the D2 protein.

D1 and D2 are structurally related proteins forming the core of the photosystem II reaction center. The two proteins have several loop regions including an extended stroma-exposed loop between transmembrane helix D and parallel helix de. This loop (the D-de loop) is phylogenetically conserved in both proteins. The role of the D-de loop in photosystem II was studied in Synechocystis sp. PCC 6803 by constructing a chimeric D2 protein in which the stroma-exposed loop of D1 replaced that of D2. In one of the transgenic lines, a single-base deletion shifted the reading frame of the chimeric gene leading to loss of D2 accumulation and photosystem II assembly. Selection for spontaneous reversion to photoautotrophy yielded several suppressor mutants, five of which were analyzed. In all, further frameshifts in the inserted loop piece restored the original reading frame allowing readthrough to the normal carboxy terminus. However, the sequences in the restored D-de loop varied widely among the mutants. Changes ranged from a deletion of one amino acid residue to an insertion of 31, while the net charge of the D-de loop increased by up to 12 units. Mutant electron transfer rates and photoautotrophic growth were only mildly affected as compared to wild type. Nevertheless, in all mutants, the hydropathy profile of the stroma-exposed D-de loop region maintained its hydrophilic character including turns in similar locations. We conclude that the stroma-exposed, D-de loop of the D2 protein can accommodate drastic composition and size changes without extensive functional consequences in photosystem II.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

Structure of the autoregulatory pseudoknot within the gene 32 messenger RNA of bacteriophages T2 and T6: a model for a possible family of structurally related RNA pseudoknots.

A 36-nucleotide RNA with a sequence corresponding to the 5' end region of the gene 32 mRNA of bacteriophages T2 and T6 was analyzed by one- and two-dimensional NMR methods. NMR results provide clear evidence that the RNA is folded into a pseudoknot structure with two coaxial stems connected by two loops, in a classic pseudoknot topology. The pseudoknot is unusual in that one of the loops consists of only one nucleotide, which spans the major groove of a seven base pair helical stem. Imino proton resonances indicate the hydrogen bonding pattern within the pseudoknot, and two-dimensional NOE spectra provide information that describes many of the structural features. The temperature dependence of the UV absorption and imino proton exchange rates provides insight into the stability of the pseudoknot. A three-dimensional model of the pseudoknot that is consistent with our NMR data is presented, and features that may be important for stabilizing the pseudoknot structure are discussed. A substantial number of other putative RNA pseudoknots described in the literature have sequences and topologies that appear to be related to the T2 and T6 pseudoknots. We propose that these RNAs may be members of a family of pseudoknots related by a similar structural motif, which we refer to as "common pseudoknot motif 1" or CPK1. The bacteriophage T2/T6 pseudoknot can be considered a structural model for the CPK1 family. The common features of the CPK1 pseudoknots are a stem 2 with six or seven base pairs, a loop 1 consisting of a single adenosine, and a variable length stem 1 and loop 2. The first "dangling" nucleotide at the 3' end of the molecule probably stabilizes stem 2. The CPK1 family includes several of the retroviral pseudoknots associated with mRNA frameshifting and readthrough. The work presented here describes the first detailed NMR analysis of an RNA pseudoknot with an entirely natural nucleotide sequence.

Bacteriophages↗
PubMed1991

Transcriptional organization and regulation of the nosiheptide resistance gene in Streptomyces actuosus.

The nosiheptide resistance gene (nshR) and a putative regulatory gene (nshA) are found together on a 2326 bp BamHI-PstI DNA fragment isolated from Streptomyces actuosus ATCC 25421. The putative regulatory gene, nshA, situated upstream from the nosiheptide resistance gene in the 2326 bp DNA fragment, contains apparent DNA-binding and RNA-binding domains. Interruption of nshA in the chromosome of S. actuosus alters nosiheptide production, suggesting that nshA is involved in regulation of nosiheptide biosynthesis. Two transcription initiation sites were found upstream of nshA as demonstrated by high-resolution S1 nuclease mapping. A weak transcription start site for nshR was found which initiated transcription from the first nucleotide of the open reading frame. Although a stem-loop structure with apparent termination activity was found between nshA and nshR, readthrough of transcription between nshA and nshR was demonstrated by S1 nuclease mapping of the 3' terminus of the nshA transcript. Time-course S1 experiments of the three promoters (nshA-pl, nshA-p2, nshR-p) indicated highly regulated differential expression of the promoters. nshA-p2 is a strong, constitutive promoter whereas 30% of the total nshA-p1/p2 transcript reads through the terminator and into the nshR gene, accounting for more than half of the total steady-state nshR transcript. The implications of the regulation of nshA and nshR gene expression, as well as the expression of two other linked genes, are presented.

Amino Acid Sequence↗
PubMed1993

Enhanced readthrough of opal (UGA) stop codons and production of Mycoplasma pneumoniae P1 epitopes in Escherichia coli.

Expression of mycoplasma sequences in Escherichia coli is often hindered by an unusual mycoplasmal codon usage pattern: the UGA stop codon is utilized for tryptophan. This may result in the truncation of cloned proteins and may prevent the detection of products of many cloned genes. To circumvent this translation barrier, we have developed an expression system for the production of mycoplasma proteins in E. coli. The efficiency of an opal suppressor tRNA (trpT176) was augmented with other suppressor mutations (prfB3 or rrsB(SuUGA-delta C1054)) which influence termination events. System efficacy was analyzed by employing suppressor mutations in the expression of TGA-containing sequences from the P1 protein-encoding gene of Mycoplasma pneumoniae.

Adhesins, Bacterial↗
PubMed1991

Characterization of the major promoter for the plasmid-encoded sucrose genes scrY, scrA, and scrB.

Sucrose genes from a Salmonella thompson plasmid were cloned in Escherichia coli K-12. A physical map and a genetic map of the genes were constructed, revealing strong homology with the scr regulon from the Salmonella typhimurium plasmid pUR400. Two promoters were examined after being subcloned into transcriptional fusion vectors. Primer extension analysis and site-directed mutagenesis were used to identify the precise location of the promoter of scrY, scrA, and scrB. Transcription from this promoter was regulated over a 1,000-fold range by the combined effects of ScrR-mediated repression and catabolite repression. A putative cyclic AMP receptor protein binding site centered 72.5 bp upstream of the start point of transcription of scrY appeared to be essential for full activity of the scrY promoter. Transcription from the putative scrK promoter was far less sensitive to repression by ScrR. In ScrR+ cells, readthrough transcription from the putative scrK promoter into scrY accounted for less than 10% of scrY expression.

Amino Acid Sequence↗
PubMed1997

Transcription-frequency-dependent modulation of an attenuator in a ribosomal protein-RNA polymerase operon requires an upstream site.

Although the attenuator located between the ribosomal protein and RNA polymerase gene domains of the Escherichia coli rplKAJLrpoBC operon has a maximum termination efficiency of 80%, the level of termination is diminished with decreasing transcription frequency. In this report, the use of transcriptional fusions to further investigate the mechanism of transcription-frequency-dependent regulation is described. The termination efficiency of two other weak terminators was assayed over a wide range of transcription frequencies programmed by different strength promoters. The results indicated that a decrease in termination efficiency with decreasing transcription frequency is not an inherent property of weak terminators. Deletion of the 165 bp segment located 439-274 bp upstream of the attenuator abrogated the difference in termination efficiency normally seen between high and low levels of transcription. This suggests that a cis-acting site located in this upstream region is necessary for transcription-frequency-dependent modulation of the attenuator's function. However, this site apparently works only in combination with the attenuator, since it did not cause transcription-frequency-dependent modulation when placed upstream of two other weak terminators. Analysis of the readthrough frequencies of single or tandem copies of the attenuator indicated that the transcription complexes which pass through the attenuator have not been converted to termination-resistant complexes in a manner analogous to the N-mediated antitermination system of lambda. Finally, an examination of termination efficiency in three nusA mutants suggested that although NusA increases readthrough at the attenuator it is not directly involved in transcription-frequency-dependent modulation.

Bacterial Proteins↗
PubMed1992

Processing of a polycistronic mRNA requires a 5' cis element and active translation.

We have characterized a major processed species of mRNA in the his operon of Salmonella typhimurium. In vivo and in vitro analyses of the his transcripts from wild-type and mutant strains using S1 nuclease protection assays, measurements of RNA stability, deletion mapping, gel retardation, and in vitro translation assays demonstrate that the distal portion of the polycistronic his mRNA is processed, resulting in increased stability. The processing event requires an upstream cis-acting element and translation of the cistron immediately downstream of the 5' end of the processed species. The cistrons contained in this segment are also independently transcribed from an internal promoter which is maximally active in the absence of readthrough transcription from the primary promoter.

Base Sequence↗
PubMed1996

In vitro characterization of mutant yeast RNA polymerase II with reduced binding for elongation factor TFIIS.

We have reported previously the isolation and genetic characterization of mutations in the gene encoding the largest subunit of yeast RNA polymerase II (RNAPII), which lead to 6-azauracil (6AU)-sensitive growth. It was suggested that these mutations affect the functional interaction between RNAPII and transcription-elongation factor TFIIS because the 6AU-sensitive phenotype of the mutant strains was similar to that of a strain defective in the production of TFIIS and can be suppressed by increasing the dosage of the yeast TFIIS-encoding gene, PPR2, RNAPIIs were purified and characterized from two independent 6AU-sensitive yeast mutants and from wild-type (wt) cells. In vitro, in the absence of TFIIS, the purified wt polymerase and the two mutant polymerases showed similar specific activity in polymerization, readthrough at intrinsic transcriptional arrest sites and nascent RNA cleavage. In contrast to the wt polymerase, both mutant polymerases were not stimulated by the addition of a 3-fold molar excess of TFIIS in assays of promoter-independent transcription, readthrough or cleavage. However, stimulation of the ability of the mutant RNAPIIs to cleave nascent RNA and to read through intrinsic arrest sites was observed at TFIIS:RNAPII molar ratios greater than 600:1. Consistent with these findings, the binding affinity of the mutant polymerases for TFIIS was found to be reduced by more than 50-fold compared with that of the wt enzyme. These studies demonstrate that TFIIS has an important role in the regulation of transcription by yeast RNAPII and identify a possible binding site for TFIIS on RNAPII.

Binding Sites↗