scholarly journals Two Regions of GerE Required for Promoter Activation in Bacillus subtilis

2002 ◽  
Vol 184 (1) ◽  
pp. 241-249 ◽  
Author(s):  
Dinene L. Crater ◽  
Charles P. Moran

ABSTRACT GerE from Bacillus subtilis is the smallest member of the LuxR-FixJ family of transcription activators. Its 74-amino-acid sequence is similar over its entire length to the DNA binding domain of this protein family, including a putative helix-turn-helix (HTH) motif. In this report, we sought to define regions of GerE involved in promoter activation. We examined the effects of single alanine substitutions at 19 positions that were predicted by the crystal structure of GerE to be located on its surface. A single substitution of alanine for the phenylalanine at position 6 of GerE (F6A) resulted in decreased transcription in vivo and in vitro from the GerE-dependent cotC promoter. However, the F6A substitution had little effect on transcription from the GerE-dependent cotX promoter. In contrast, a single alanine substitution for the leucine at position 67 (L67A) reduced transcription from the cotX promoter, but not from the cotC promoter. The results of DNase I protection assays and in vitro transcription reactions lead us to suggest that the F6A and L67A substitutions define two regions of GerE, activation region 1 (AR1) and AR2, that are required for activation of the cotC and cotX promoters, respectively. A comparison of our results with those from studies of MalT and BvgA indicated that other members of the LuxR-FixJ family may use more than one surface to interact with RNA polymerase during promoter activation.

1977 ◽  
Author(s):  
F.J. Morgan ◽  
G.S. Begg ◽  
C.N. Chesterman

The amino acid sequence of human platelet factor 4 (PF4) has been studied. PF4 is a platelet specific protein with antiheparin activity, released from platelets as a proteoglycan complex, whose measurement may provide an important index of platelet activation both in vivo and in vitro. These studies were undertaken to characterize fully the PF4 molecule. PF4 is a stable tetramer, composed of identical subunits, each with a molecular weight based on the sequence studies of approx. 7,770. Each PF4 subunit contains 69 amino acids, including 4 half-cystine (# 10, 12, 36, 37), one tyrosine (# 59), 3 arginine and 8 lysine, but no methionine, phenylalanine or tryptophan residues. The basic residues are predominantly in the C-terminal region. The tryptic peptides were aligned after studies which included tryptic digestion of citraconylated RCM-PF4, and automated Edman degradation of RCM-PF4 and citraconylated tryptic peptides. No glycopeptides were detected. This structural information should enable clear distinction to be made between PF4 and other platelet proteins such as β thromboglobulin. The provisional amino acid sequence of each subunit is:Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val-Lys-Thr-Thr-Ser-Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Cys-Pro-Thr-Ala-Gln-Ile-Leu-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Pro-Leu-Asp-Leu-Gln-Ala-Tyr-Leu-Lys-Ile-Lys(Lys, Lys, Ser, Glx, Leu, Leu)


2001 ◽  
Vol 183 (6) ◽  
pp. 1921-1927 ◽  
Author(s):  
Jian Qiu ◽  
John D. Helmann

ABSTRACT Transcriptional selectivity derives, in large part, from the sequence-specific DNA-binding properties of the ς subunit of RNA polymerase. There are 17 ς factors in Bacillus subtilis which, in general, recognize distinct sets of promoters. However, some ς factors have overlapping promoter selectivity. We hypothesize that the overlap between the regulons activated by the ςX and ςW factors can be explained by overlapping specificity for the −10 region: ςX recognizes −10 elements with the sequence CGAC and ςW recognizes CGTA, while both can potentially recognize CGTC. To test this model, we mutated the ςX-specific autoregulatory site (PX), containing the −10 element CGAC, to either CGTC or GCTA. Conversely, the ςW autoregulatory site (PW) was altered from CGTA to CGTC or CGAC. Transcriptional analyses, both in vitro and in vivo, indicate that changes to the −10 element are sufficient to switch a promoter from the ςX to the ςW regulon or, conversely, from the ςW to the ςX regulon, but context effects clearly play an important role in determining promoter strength. It seems likely that these subtle differences in promoter selectivity derive from amino acid differences in conserved region 2 of ς, which contacts the −10 element. However, we were unable to alter promoter selectivity by replacements of two candidate recognition residues in ςW.


1995 ◽  
Vol 15 (3) ◽  
pp. 1467-1478 ◽  
Author(s):  
S A Shaaban ◽  
B M Krupp ◽  
B D Hall

In order to identify catalytically important amino acid changes within the second-largest subunit of yeast RNA polymerase III, we mutagenized selected regions of its gene (RET1) and devised in vivo assays for both increased and decreased transcription termination by this enzyme. Using as the reporter gene a mutant SUP4-o tRNA gene that in one case terminates prematurely and in the other case fails to terminate, we screened mutagenized RET1 libraries for reduced and increased transcription termination, respectively. The gain in suppression phenotype was in both cases scored as a reduction in the accumulation of red pigment in yeast strains harboring the ade2-1 ochre mutation. Termination-altering mutations were obtained in regions of the RET1 gene encoding amino acids 300 to 325, 455 to 486, 487 to 521, and 1061 to 1082 of the protein. In degree of amino acid sequence conservation, these range from highly variable in the first to highly conserved in the last two regions. Residues 300 to 325 yielded mainly reduced-termination mutants, while in region 1061 to 1082, increased-termination mutants were obtained exclusively. All mutants recovered, while causing gain of suppression with one SUP4 allele, brought about a reduction in suppression with the other allele, thus confirming that the phenotype is due to altered termination rather than an elevated level of transcription initiation. In vitro transcription reactions performed with extracts from several strong mutants demonstrated that the mutant polymerases respond to RNA terminator sequences in a manner that matches their in vivo termination phenotypes.


Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2796-2806 ◽  
Author(s):  
Vivienne Mahon ◽  
Cyril J. Smyth ◽  
Stephen G. J. Smith

The pathogenesis of diarrhoeal disease due to human enterotoxigenic Escherichia coli absolutely requires the expression of fimbriae. The expression of CS1 fimbriae is positively regulated by the AraC-like protein Rns. AraC-like proteins are DNA-binding proteins that typically contain two helix–turn–helix (HTH) motifs. A program of pentapeptide insertion mutagenesis of the Rns protein was performed, and this revealed that both HTH motifs are required by Rns to positively regulate CS1 fimbrial gene expression. Intriguingly, a pentapeptide insertion after amino acid C102 reduced the ability of Rns to transactivate CS1 fimbrial expression. The structure of Rns in this vicinity (NACRS) was predicted to be disordered and thus might act as a flexible linker. This hypothesis was confirmed by deletion of this amino acid sequence from the Rns protein; a truncated protein that lacked this sequence was no longer functional. Strikingly, this sequence could be functionally substituted in vivo and in vitro by a flexible seven amino acid sequence from another E. coli AraC-like protein RhaS. Our data indicate that HTH motifs and a flexible sequence are required by Rns for maximal activation of fimbrial gene expression.


Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 828 ◽  
Author(s):  
Jochen M. Wettengel ◽  
Benjamin J. Burwitz

Hepatitis B is a major global health problem, with an estimated 257 million chronically infected patients and almost 1 million deaths per year. The causative agent is hepatitis B virus (HBV), a small, enveloped, partially double-stranded DNA virus. HBV has a strict species specificity, naturally infecting only humans and chimpanzees. Sodium taurocholate co-transporting polypeptide (NTCP), a bile acid transporter expressed on hepatocytes, has been shown to be one of the key factors in HBV infection, playing a crucial role in the HBV entry process in vitro and in vivo. Variations in the amino acid sequence of NTCP can inhibit HBV infection and, therefore, contributes, in part, to the species barrier. This discovery has revolutionized the search for novel animal models of HBV. Indeed, it was recently shown that variations in the amino acid sequence of NTCP represent the sole species barrier for HBV infection in macaques. Here, we review what is known about HBV entry through the NTCP receptor and highlight how this knowledge has been harnessed to build new animal models for the study of HBV pathogenesis and curative therapies.


1989 ◽  
Vol 9 (5) ◽  
pp. 1987-1995
Author(s):  
A A Amin ◽  
P D Sadowski

We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein. The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP. Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination. Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity. Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity. On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity. Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95%. In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination. On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain. The possible reasons for this result are discussed.


1998 ◽  
Vol 334 (3) ◽  
pp. 659-667 ◽  
Author(s):  
Christine RASCHER ◽  
Andreas PAHL ◽  
Anja PECHT ◽  
Kay BRUNE ◽  
Werner SOLBACH ◽  
...  

The immunosuppressive effects of the fungal metabolite cyclosporin A (CsA) are mediated primarily by binding to cyclophilins (Cyps). The resulting CsA–Cyp complex inhibits the Ca2+-regulated protein phosphatase calcineurin and down-regulates signal transduction events. Previously we reported that CsA is a potent inhibitor of infections transmitted by the human pathogenic protozoan parasite Leishmania major in vitro and in vivo, but does not effect the extracellular growth of L. major itself. It is unknown how L. major exerts this resistance to CsA. Here we report that a major Cyp, besides additional isoforms with the same N-terminal amino acid sequence, was expressed in L. major. The cloned and sequenced gene encodes a putative 174-residue protein called L. major Cyp 19 (LmCyp19). The recombinant LmCyp19 exhibits peptidyl-prolyl cis/trans isomerase activity with a substrate specificity and an inhibition by CsA that are characteristic of other eukaryotic Cyps. To determine whether calcineurin is involved in the discrimination of the effects of CsA we also examined the presence of a parasitic calcineurin and tested the interaction with Cyps. Despite the expression of functionally active calcineurin by L. major, neither LmCyp19 nor other L. major Cyps bound to its own or mammalian calcineurin. The amino acid sequence of most Cyps includes an essential arginine residue around the calcineurin-docking side. In LmCyp19 this is replaced by an asparagine residue. This exchange and additional charged residues are apparently responsible for the lack of LmCyp19 interaction with calcineurin. These observations indicate that resistance of L. major to CsA in vitro is mediated by the lack of complex formation with calcineurin despite CsA binding by parasitic Cyp.


2018 ◽  
Vol 1 (5) ◽  
pp. e201800148 ◽  
Author(s):  
Britta Seip ◽  
Guénaël Sacheau ◽  
Denis Dupuy ◽  
C Axel Innis

Although it is known that the amino acid sequence of a nascent polypeptide can impact its rate of translation, dedicated tools to systematically investigate this process are lacking. Here, we present high-throughput inverse toeprinting, a method to identify peptide-encoding transcripts that induce ribosomal stalling in vitro. Unlike ribosome profiling, inverse toeprinting protects the entire coding region upstream of a stalled ribosome, making it possible to work with random or focused transcript libraries that efficiently sample the sequence space. We used inverse toeprinting to characterize the stalling landscapes of free and drug-boundEscherichia coliribosomes, obtaining a comprehensive list of arrest motifs that were validated in vivo, along with a quantitative measure of their pause strength. Thanks to the modest sequencing depth and small amounts of material required, inverse toeprinting provides a highly scalable and versatile tool to study sequence-dependent translational processes.


1996 ◽  
Vol 16 (11) ◽  
pp. 6313-6324 ◽  
Author(s):  
I M Wang ◽  
J C Blanco ◽  
S Y Tsai ◽  
M J Tsai ◽  
K Ozato

Interferon regulatory factors (IRFs) bind to the interferon-stimulated response element (ISRE) and regulate interferon- and virus-mediated gene expression. IRF-1 acts as a transcriptional activator, while IRF-2 acts as a repressor. Here we show that IRF-1 and IRF-2 bind to both cellular TFIIB, a component of the basal transcription machinery, and recombinant TFIIB (rTFIIB) and that this protein-protein interaction facilitates binding of IRFs to the ISRE. A functional interaction between TFIIB and IRF was assessed by a newly established in vitro transcription assay in which recombinant IRF-1 (rIRF-1) stimulated transcription specifically from an ISRE-containing template. With this assay we show that rIRF-1 and rTFIIB cooperatively enhance the ISRE promoter in vitro. We found that the activity of an ISRE-containing promoter was cooperatively enhanced upon cotransfection of TFIIB and IRF-1 cDNAs into P19 embryonal carcinoma cells, further demonstrating functional interactions in vivo. The cooperative enhancement by TFIIB and IRF-1 was independent of the TATA sequence in the ISRE promoter but dependent on the initiator sequence (Inr) and was abolished when P19 cells were induced to differentiate by retinoic acid treatment. In contrast, cotransfection of TFIIB and IRF-1 into NIH 3T3 cells resulted in a dose-dependent repression of promoter activation which occurred in a TATA-dependent manner. Our results indicate the presence of a cell type-specific factor that mediates the functional interaction between IRFs and TFIIB and that acts in conjunction with the requirement of TATA and Inr for promoter activation.


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