scholarly journals Phage T5 two-step injection

2019 ◽  
Author(s):  
John Davison
Keyword(s):  
Rolling Circle ◽  
Cis Acting ◽  
Dnaa Protein ◽  
Dna Injection ◽  
Genome Expression ◽  
Expression Of Genes ◽  
Membrane Bound ◽  
A Site ◽  
Bacteriophage T5 ◽  
Circle Formation

AbstractEscherichia coli bacteriophage T5 differs from most phages in that it injects its genome in two steps: First Step Transfer, FST, corresponding to leftmost 7.9% of the genome and Second Step Transfer, SST, corresponding to the remainder of the genome. Expression of genes A1 and A2 is required for SST. DNA injection stops at a site known as the injection stop signal (iss) which is a cis acting site located in the large untranslated region of the Left Terminal Repeat (LTR). The iss region is extremely complicated with many repeats, inverted repeats and palindromes. This report compares the iss regions of 25 T5-like phages and shows that all have a common conserved structure including a series of 8 DnaA boxes arranged in a highly specific manner; reminiscent of the origin of replication (oriC) of E. coli. DnaA protein, which binds to DnaA boxes, is a mostly membrane bound. A new, radically different, mechanism of T5 2-step injection is proposed whereby injecting T5 DNA stops at the plasma membrane due to the binding of the iss DnaA boxes to membrane-bound DnaA protein. Injection of the SST continues later via the combined action of the A1 and A2 proteins which cleave the FST DNA at a site upstream (right) of the iss region, thereby liberating it. They also cleave the incoming SST DNA at the same site on the RTR thus facilitating circularisation of one complete genome via the cohesive ends. Circle formation protects the T5 DNA from the degradative action of the RecBCD nuclease and eventually leads to rolling circle DNA replication.

scholarly journals Membrane-Bound Nitrate Reductase Is Required for Anaerobic Growth in Cystic Fibrosis Sputum

2007 ◽  
Vol 189 (12) ◽  
pp. 4449-4455 ◽  
Author(s):  
Kelli L. Palmer ◽  
Stacie A. Brown ◽  
Marvin Whiteley
Keyword(s):  
Cystic Fibrosis ◽  
Nitrate Reductase ◽  
Mutational Analysis ◽  
Autosomal Recessive Disorder ◽  
Opportunistic Pathogen ◽  
Anaerobic Growth ◽  
Expression Of Genes ◽  
Membrane Bound ◽  
Lung Infections

ABSTRACT The autosomal recessive disorder cystic fibrosis (CF) affects approximately 70,000 people worldwide and is characterized by chronic bacterial lung infections with the opportunistic pathogen Pseudomonas aeruginosa. To form a chronic CF lung infection, P. aeruginosa must grow and proliferate within the CF lung, and the highly viscous sputum within the CF lung provides a likely growth substrate. Recent evidence indicates that anaerobic microenvironments may be present in the CF lung sputum layer. Since anaerobic growth significantly enhances P. aeruginosa biofilm formation and antibiotic resistance, it is important to examine P. aeruginosa physiology and metabolism in anaerobic environments. Measurement of nitrate levels revealed that CF sputum contains sufficient nitrate to support significant P. aeruginosa growth anaerobically, and mutational analysis revealed that the membrane-bound nitrate reductase is essential for P. aeruginosa anaerobic growth in an in vitro CF sputum medium. In addition, expression of genes coding for the membrane-bound nitrate reductase complex is responsive to CF sputum nitrate levels. These findings suggest that the membrane-bound nitrate reductase is critical for P. aeruginosa anaerobic growth with nitrate in the CF lung.

scholarly journals Posttranslational modification and intracellular transport of a trypanosome variant surface glycoprotein.

1986 ◽  
Vol 103 (1) ◽  
pp. 255-263 ◽  
Author(s):  
J D Bangs ◽  
N W Andrews ◽  
G W Hart ◽  
P T Englund
Keyword(s):  
Cell Surface ◽  
Phospholipase C ◽  
Intracellular Transport ◽  
Signal Sequence ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Hydrophobic Peptide ◽  
Membrane Bound ◽  
A Site ◽  
Kinetics Of

After synthesis on membrane-bound ribosomes, the variant surface glycoprotein (VSG) of Trypanosoma brucei is modified by: (a) removal of an N-terminal signal sequence, (b) addition of N-linked oligosaccharides, and (c) replacement of a C-terminal hydrophobic peptide with a complex glycolipid that serves as a membrane anchor. Based on pulse-chase experiments with the variant ILTat-1.3, we now report the kinetics of three subsequent processing reactions. These are: (a) conversion of newly synthesized 56/58-kD polypeptides to mature 59-kD VSG, (b) transport to the cell surface, and (c) transport to a site where VSG is susceptible to endogenous membrane-bound phospholipase C. We found that the t 1/2 of all three of these processes is approximately 15 min. The comparable kinetics of these processes is compatible with the hypotheses that transport of VSG from the site of maturation to the cell surface is rapid and that VSG may not reach a phospholipase C-containing membrane until it arrives on the cell surface. Neither tunicamycin nor monensin blocks transport of VSG, but monensin completely inhibits conversion of 58-kD VSG to the mature 59-kD form. In the presence of tunicamycin, VSG is synthesized as a 54-kD polypeptide that is subsequently processed to a form with a slightly higher Mr. This tunicamycin-resistant processing suggests that modifications unrelated to N-linked oligosaccharides occur. Surprisingly, the rate of VSG transport is reduced, but not abolished, by dropping the chase temperature to as low as 10 degrees C.

scholarly journals Sequences regulating temporal poly(A) site switching in the adenovirus major late transcription unit.

1991 ◽  
Vol 11 (12) ◽  
pp. 5977-5984 ◽  
Author(s):  
J D DeZazzo ◽  
E Falck-Pedersen ◽  
M J Imperiale
Keyword(s):  
Regulatory Element ◽  
Transcription Unit ◽  
Late Infection ◽  
Cis Acting ◽  
Temporal Regulation ◽  
Deletion Mutagenesis ◽  
Late Transcription ◽  
A Site ◽  
Adenovirus Recombinant ◽  
Selection Of

Temporal regulation of poly(A) site choice occurs in an adenovirus recombinant encoding a miniature version of the major late transcription unit with two poly(A) sites, L1 and L3. Using deletion mutagenesis, we have looked directly for cis-acting elements regulating poly(A) site choice in this recombinant. From this work, we draw two main conclusions. First, elements other than the AAUAAA and downstream sequences of the L1 poly(A) site are required for temporal regulation of poly(A) site choice during infection. Second, these regions function in two distinct modes during infection. The two regions enhance selection of the L1 poly(A) site in an additive manner during an early infection, but deletion of either element abolishes the switch in poly(A) site choice during a late infection. This work documents the first example of a regulatory element downstream of a core poly(A) region.

Role of the potassium/lysine cationic center in catalysis and functional asymmetry in membrane-bound pyrophosphatases

2018 ◽  
Vol 475 (6) ◽  
pp. 1141-1158 ◽  
Author(s):  
Erika Artukka ◽  
Heidi H. Luoto ◽  
Alexander A. Baykov ◽  
Reijo Lahti ◽  
Anssi M. Malinen
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Systematic Analysis ◽  
Change Mechanism ◽  
Excess Substrate ◽  
Na Ions ◽  
Membrane Bound ◽  
A Site ◽  
Pyrophosphate Hydrolysis

Membrane-bound pyrophosphatases (mPPases), which couple pyrophosphate hydrolysis to transmembrane transport of H+ and/or Na+ ions, are divided into K+,Na+-independent, Na+-regulated, and K+-dependent families. The first two families include H+-transporting mPPases (H+-PPases), whereas the last family comprises one Na+-transporting, two Na+- and H+-transporting subfamilies (Na+-PPases and Na+,H+-PPases, respectively), and three H+-transporting subfamilies. Earlier studies of the few available model mPPases suggested that K+ binds to a site located adjacent to the pyrophosphate-binding site, but is substituted by the ε-amino group of an evolutionarily acquired lysine residue in the K+-independent mPPases. Here, we performed a systematic analysis of the K+/Lys cationic center across all mPPase subfamilies. An Ala → Lys replacement in K+-dependent mPPases abolished the K+ dependence of hydrolysis and transport activities and decreased these activities close to the level (4–7%) observed for wild-type enzymes in the absence of monovalent cations. In contrast, a Lys → Ala replacement in K+,Na+-independent mPPases conferred partial K+ dependence on the enzyme by unmasking an otherwise conserved K+-binding site. Na+ could partially replace K+ as an activator of K+-dependent mPPases and the Lys → Ala variants of K+,Na+-independent mPPases. Finally, we found that all mPPases were inhibited by excess substrate, suggesting strong negative co-operativity of active site functioning in these homodimeric enzymes; moreover, the K+/Lys center was identified as part of the mechanism underlying this effect. These findings suggest that the mPPase homodimer possesses an asymmetry of active site performance that may be an ancient prototype of the rotational binding-change mechanism of F-type ATPases.

scholarly journals Genome-wide association mapping of transcriptome variation in Mimulus guttatus indicates differing patterns of selection on cis- versus trans-acting mutations

2021 ◽  
Author(s):  
Keely Brown ◽  
John K. Kelly
Keyword(s):  
Allele Frequency ◽  
Plant Traits ◽  
Balancing Selection ◽  
Strong Predictor ◽  
Purifying Selection ◽  
Striking Difference ◽  
Nucleotide Polymorphisms ◽  
Mimulus Guttatus ◽  
Cis Acting ◽  
Expression Of Genes

We measured the floral bud transcriptome of 151 fully sequenced lines of Mimulus guttatus from one natural population. Thousands of single nucleotide polymorphisms (SNPs) are implicated as transcription regulators, but there is a striking difference in the Allele Frequency Spectrum (AFS) of cis-acting and trans-acting mutations. Cis-SNPs have intermediate frequencies (consistent with balancing selection) while trans-SNPs exhibit a rare-alleles model (consistent with purifying selection). This pattern only becomes clear when transcript variation is normalized on a gene-to-gene basis. If a global normalization is applied, as is typically in RNAseq experiments, asymmetric transcript distributions combined with rarity disequilibrium produce a super-abundance of false positives for trans-acting SNPs. To explore the cause of purifying selection on trans-acting mutations, we identified gene expression modules as sets of co-expressed genes. The extent to which trans-acting mutations influence modules is a strong predictor of allele frequency. Mutations altering expression of genes with high connectedness (those that are highly predictive of the representative module expression value) have the lowest allele frequency. The expression modules can also predict whole-plant traits such as flower size. We find that a substantial portion of the genetic (co)variance among traits can be described as an emergent property of genetic effects on expression modules.

scholarly journals Structural basis of adenylyl cyclase 9 activation

2021 ◽  
Author(s):  
Chao Qi ◽  
Pia Lavriha ◽  
Ved Mehta ◽  
Basavraj Khanppnavar ◽  
Inayathulla Mohammed ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Binding Site ◽  
Adenylyl Cyclase ◽  
Conformational Changes ◽  
Structural Basis ◽  
Structural Studies ◽  
Nucleotide Analogue ◽  
C Terminus ◽  
Membrane Bound ◽  
A Site

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four new conformations show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

scholarly journals Chromatin organization of the Saccharomyces cerevisiae 2 microns plasmid depends on plasmid-encoded products.

1985 ◽  
Vol 5 (9) ◽  
pp. 2190-2196 ◽  
Author(s):  
B E Veit ◽  
W L Fangman
Keyword(s):  
Copy Number ◽  
Plasmid Copy Number ◽  
Chromatin Organization ◽  
Open Reading Frames ◽  
Plasmid Copy ◽  
Cis Acting ◽  
Stable Plasmid ◽  
2 Micron Plasmid ◽  
Plasmid Genes ◽  
A Site

We have used gene disruptions and nuclease probes to assess the roles of yeast 2 micron plasmid genes in plasmid chromatin organization. The chromatin structure at the replication origin is not dependent on any of the four major open reading frames, A, B, C, or D. While stable plasmid maintenance is known to depend on a cis-acting locus STB and genes B and C, we find that only gene B influences STB chromatin. Other interactions between plasmid gene products and sequences may reflect gene regulation: the chromatin organization at the 5' end of gene A, which codes for a site-specific recombinase, depends on both gene B and gene C. Since disruption of gene C results in an increase in plasmid copy number that is dependent on gene A, we propose that gene C (and probably gene B) control copy number by regulating the level of the gene A recombinase.

scholarly journals Improvement of bovine in vitro embryo production by vitamin K2 supplementation

Reproduction ◽  
2014 ◽  
Vol 148 (5) ◽  
pp. 489-497 ◽  
Author(s):  
Luis Manuel Baldoceda-Baldeon ◽  
Dominic Gagné ◽  
Christian Vigneault ◽  
Patrick Blondin ◽  
Claude Robert
Keyword(s):  
Vitamin K ◽  
Mitochondrial Function ◽  
Blastocyst Stage ◽  
Mitochondrial Activity ◽  
Bovine Embryos ◽  
Current Standard ◽  
Expression Of Genes ◽  
Mammalian Embryos ◽  
Membrane Bound

Mitochondria play an important role during early development in mammalian embryos. It has been shown that properly controlled follicular preparation increases the likelihood ofin-vitro-produced bovine embryos reaching the blastocyst stage and that competent embryos exhibit heightened expression of genes associated with mitochondrial function. We hypothesized that apparently incompetent embryos could be rescued by restoring mitochondrial function. It has been shown that vitamin K2(a membrane-bound electron carrier similar to ubiquinone) can restore mitochondrial dysfunction in eukaryotic cells. The aim of this study was therefore to investigate the effects of vitamin K2on bovine embryonic developmentin vitro. The vitamin was found most effective when added 72 h after fertilization. It produced a significant (P<0.05) increase in the percentage of blastocysts (+8.6%), more expanded blastocysts (+7.8%), and embryos of better morphological quality. It improved the mitochondrial activity significantly and had a measurable impact on gene expression. This is the first demonstration that current standard conditions ofin vitroproduction of bovine embryos may be inadequate due to the lack of support for mitochondrial function and may be improved significantly by supplementing the culture medium with vitamin K2.

scholarly journals Characterization of the polyomavirus late polyadenylation signal.

1995 ◽  
Vol 15 (9) ◽  
pp. 4783-4790 ◽  
Author(s):  
D B Batt ◽  
G G Carmichael
Keyword(s):  
Late Phase ◽  
Rnase Protection Assay ◽  
Polyadenylation Signal ◽  
Cis Elements ◽  
Rnase Protection ◽  
Upstream Site ◽  
Cis Acting ◽  
A Site

The polyomavirus late polyadenylation signal is used inefficiently during the late phase of a productive viral infection. Inefficient polyadenylation serves an important purpose for viral propagation, as it allows a splicing event that stabilizes late transcripts (G. R. Adami, C. W. Marlor, N. L. Barrett, and G. G. Carmichale, J. Virol. 63:85-93, 1989; R. P. Hyde-DeRuyscher and G. G. Carmichael, J. Virol. 64:5823-5832, 1990). We have recently shown that late-strand readthrough transcripts serve as natural antisense molecules to downregulate early-strand RNA levels at late times in infection (Z. Liu, D. B. Batt, and G. G. Carmichael, Proc. Natl. Acad. Sci. USA 91:4258-4262, 1994). Thus, poor polyadenylation contributes to the early-late switch by allowing the formation of more stable late RNAs and by forming antisense RNA to early RNAs. The importance of late poly(A) site inefficiency in the viral life cycle has prompted us to map the cis elements of this site. Since the polyomavirus late site proved a poor substrate for in vitro polyadenylation, we used an in vivo assay which allowed us to map the cis sequences required for its function. In this assay, various fragments containing the AAUAAA and different surrounding sequences were placed 1.4 kb upstream of a second, wild-type signal. The second signal served to stabilize transcripts that are not processed at the upstream site, allowing accurate quantitation of relative poly(A) site use by an RNase protection assay. Processing was primary at the upstream site when a large fragment surrounding the poly(A) signal (50 nucleotides [nt] upstream and 90 nt downstream) was tested in this assay, demonstrating that this fragment contains the essential cis elements. Deletion analysis of this fragment revealed that most but not all upstream sequences can be removed with little effect on polyadenylation efficiency, indicating the absence of a strong stimulatory upstream element. Deletion of all but 25 nt downstream of the AAUAAA reduced polyadenylation activity only by half, demonstrating that processing can occur at this site despite the lack of downstream sequences. Thus, the core cis element for polyadenylation is quite small, with most important cis-acting elements lying within 19 nt upstream and 25 nt downstream of the AAUAAA sequence. This core contains the AAUAAA hexanucleotide, an upstream A/U-rich element, and three identical repeats of a 6-nt sequence, UAUUCA. Polyadenylation was eliminated or greatly reduced when either the AAUAAA or the three repeats were mutated.

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