scholarly journals A novel mitogen-inducible gene product related to p50/p105-NF-kappa B participates in transactivation through a kappa B site.

1992 ◽  
Vol 12 (2) ◽  
pp. 685-695 ◽  
Author(s):  
V Bours ◽  
P R Burd ◽  
K Brown ◽  
J Villalobos ◽  
S Park ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Peripheral Blood ◽  
Carcinoma Cells ◽  
Nf Kappa B ◽  
Reading Frame ◽  
Amino Terminal ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Inducible Gene

A Rel-related, mitogen-inducible, kappa B-binding protein has been cloned as an immediate-early activation gene of human peripheral blood T cells. The cDNA has an open reading frame of 900 amino acids capable of encoding a 97-kDa protein. This protein is most similar to the 105-kDa precursor polypeptide of p50-NF-kappa B. Like the 105-kDa precursor, it contains an amino-terminal Rel-related domain of about 300 amino acids and a carboxy-terminal domain containing six full cell cycle or ankyrin repeats. In vitro-translated proteins, truncated downstream of the Rel domain and excluding the repeats, bind kappa B sites. We refer to the kappa B-binding, truncated protein as p50B by analogy with p50-NF-kappa B and to the full-length protein as p97. p50B is able to form heteromeric kappa B-binding complexes with RelB, as well as with p65 and p50, the two subunits of NF-kappa B. Transient-transfection experiments in embryonal carcinoma cells demonstrate a functional cooperation between p50B and RelB or p65 in transactivation of a reporter plasmid dependent on a kappa B site. The data imply the existence of a complex family of NF-kappa B-like transcription factors.

A novel mitogen-inducible gene product related to p50/p105-NF-kappa B participates in transactivation through a kappa B site

1992 ◽  
Vol 12 (2) ◽  
pp. 685-695
Author(s):  
V Bours ◽  
P R Burd ◽  
K Brown ◽  
J Villalobos ◽  
S Park ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Peripheral Blood ◽  
Carcinoma Cells ◽  
Nf Kappa B ◽  
Reading Frame ◽  
Amino Terminal ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Inducible Gene

A Rel-related, mitogen-inducible, kappa B-binding protein has been cloned as an immediate-early activation gene of human peripheral blood T cells. The cDNA has an open reading frame of 900 amino acids capable of encoding a 97-kDa protein. This protein is most similar to the 105-kDa precursor polypeptide of p50-NF-kappa B. Like the 105-kDa precursor, it contains an amino-terminal Rel-related domain of about 300 amino acids and a carboxy-terminal domain containing six full cell cycle or ankyrin repeats. In vitro-translated proteins, truncated downstream of the Rel domain and excluding the repeats, bind kappa B sites. We refer to the kappa B-binding, truncated protein as p50B by analogy with p50-NF-kappa B and to the full-length protein as p97. p50B is able to form heteromeric kappa B-binding complexes with RelB, as well as with p65 and p50, the two subunits of NF-kappa B. Transient-transfection experiments in embryonal carcinoma cells demonstrate a functional cooperation between p50B and RelB or p65 in transactivation of a reporter plasmid dependent on a kappa B site. The data imply the existence of a complex family of NF-kappa B-like transcription factors.

scholarly journals Negative Regulation of DNA Replication by the Retinoblastoma Protein Is Mediated by Its Association with MCM7

1998 ◽  
Vol 18 (5) ◽  
pp. 2748-2757 ◽  
Author(s):  
Jacqueline M. Sterner ◽  
Susan Dew-Knight ◽  
Christine Musahl ◽  
Sally Kornbluth ◽  
Jonathan M. Horowitz
Keyword(s):  
Amino Acids ◽  
Dna Replication ◽  
Protein Complexes ◽  
Replication Assay ◽  
Amino Terminal ◽  
Licensing Factor ◽  
Human Proteins ◽  
Carboxy Terminal ◽  
Related Proteins

ABSTRACT A yeast two-hybrid screen was employed to identify human proteins that specifically bind the amino-terminal 400 amino acids of the retinoblastoma (Rb) protein. Two independent cDNAs resulting from this screen were found to encode the carboxy-terminal 137 amino acids of MCM7, a member of a family of proteins that comprise replication licensing factor. Full-length Rb and MCM7 form protein complexes in vitro, and the amino termini of two Rb-related proteins, p107 and p130, also bind MCM7. Protein complexes between Rb and MCM7 were also detected in anti-Rb immunoprecipitates prepared from human cells. The amino-termini of Rb and p130 strongly inhibited DNA replication in an MCM7-dependent fashion in a Xenopus in vitro DNA replication assay system. These data provide the first evidence that Rb and Rb-related proteins can directly regulate DNA replication and that components of licensing factor are targets of the products of tumor suppressor genes.

scholarly journals Mouse Hepatitis Virus 3C-Like Protease Cleaves a 22-Kilodalton Protein from the Open Reading Frame 1a Polyprotein in Virus-Infected Cells and In Vitro

1998 ◽  
Vol 72 (3) ◽  
pp. 2265-2271 ◽  
Author(s):  
Xiao Tao Lu ◽  
Amy C. Sims ◽  
Mark R. Denison
Keyword(s):  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Open Reading Frame ◽  
Cleavage Sites ◽  
Reading Frame ◽  
Amino Terminal ◽  
Infected Cells ◽  
Carboxy Terminal ◽  
A Site

ABSTRACT The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.

scholarly journals Functional Domains of Yeast Plasmid-Encoded Rep Proteins

2001 ◽  
Vol 183 (7) ◽  
pp. 2306-2315 ◽  
Author(s):  
A. Sengupta ◽  
K. Blomqvist ◽  
A. J. Pickett ◽  
Y. Zhang ◽  
J. S. K. Chew ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Interaction ◽  
Yeast Plasmid ◽  
Amino Terminal ◽  
Plasmid Segregation ◽  
Terminal Domain ◽  
Rep Proteins ◽  
Self Association ◽  
Carboxy Terminal

ABSTRACT Both of the Saccharomyces cerevisiae 2μm circle-encoded Rep1 and Rep2 proteins are required for efficient distribution of the plasmid to daughter cells during cellular division. In this study two-hybrid and in vitro protein interaction assays demonstrate that the first 129 amino acids of Rep1 are sufficient for self-association and for interaction with Rep2. Deletion of the first 76 amino acids of Rep1 abolished the Rep1-Rep2 interaction but still allowed some self-association, suggesting that different but overlapping domains specify these interactions. Amino- or carboxy-terminally truncated Rep1 fusion proteins were unable to complement defective segregation of a 2μm-based stability vector withrep1 deleted, supporting the idea of the requirement of Rep protein interaction for plasmid segregation but indicating a separate required function for the carboxy-terminal portion of Rep1. The results of in vitro baiting assays suggest that Rep2 contains two nonoverlapping domains, both of which are capable of mediating Rep2 self-association. The amino-terminal domain interacts with Rep1, while the carboxy-terminal domain was shown by Southwestern analysis to have DNA-binding activity. The overlapping Rep1 and Rep2 interaction domains in Rep1, and the ability of Rep2 to interact with Rep1, Rep2, and DNA, suggest a model in which the Rep proteins polymerize along the 2μm circle plasmid stability locus, forming a structure that mediates plasmid segregation. In this model, competition between Rep1 and Rep2 for association with Rep1 determines the formation or disassembly of the segregation complex.

scholarly journals Two domains of p53 interact with the TATA-binding protein, and the adenovirus 13S E1A protein disrupts the association, relieving p53-mediated transcriptional repression.

1995 ◽  
Vol 15 (1) ◽  
pp. 227-234 ◽  
Author(s):  
N Horikoshi ◽  
A Usheva ◽  
J Chen ◽  
A J Levine ◽  
R Weinmann ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Binding Protein ◽  
Direct Interaction ◽  
Tata Binding Protein ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal

The tumor suppressor gene product p53 can activate and repress transcription. Both transcriptional activation and repression are thought to involve the direct interaction of p53 with the basal transcriptional machinery. Previous work has demonstrated an in vitro interaction between p53 and the TATA-binding protein that requires amino acids 20 to 57 of p53 and amino acids 220 to 271 of the TATA-binding protein. The present results show that a 75-amino-acid segment from the carboxy terminus of p53 also can bind to the TATA-binding protein in vitro, and this interaction requires amino acids 217 to 268 of the TATA-binding protein, essentially the same domain that is required for interaction with the amino-terminal domain of p53. A carboxy-terminal segment of p53 can mediate repression when bound to DNA as a GAL4-p53 fusion protein. The amino- and carboxy-terminal p53 interactions occur within the domain on the TATA-binding protein to which the adenovirus 13S E1A oncoprotein has previously been shown to bind. The 13S E1A oncoprotein can dissociate the complex formed between the carboxy-terminal domain of p53 and the TATA-binding protein and relieve p53-mediated transcriptional repression. These results demonstrate that two independent domains of p53 can potentially interact with the TATA-binding protein, and they define a mechanism--relief of repression--by which the 13S E1A oncoprotein can activate transcription through the TATA motif.

scholarly journals Regions of the Varicella-Zoster Virus Open Reading Frame 63 Latency-Associated Protein Important for Replication In Vitro Are Also Critical for Efficient Establishment of Latency

2005 ◽  
Vol 79 (8) ◽  
pp. 5069-5077 ◽  
Author(s):  
Jeffrey I. Cohen ◽  
Tammy Krogmann ◽  
Sebastien Bontems ◽  
Catherine Sadzot-Delvaux ◽  
Lesley Pesnicak
Keyword(s):  
Amino Acids ◽  
Varicella Zoster Virus ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Localization Signal ◽  
Carboxy Terminal

ABSTRACT Varicella-zoster virus (VZV) open reading frame 63 (ORF63) is one of the most abundant transcripts expressed during VZV latency in humans, and ORF63 protein has been detected in human ganglia by several laboratories. Deletion of over 90% of the ORF63 gene showed that the protein is required for efficient establishment of latency in rodents. We have constructed viruses with a series of mutations in ORF63. While prior experiments showed that transfection of cells with a plasmid expressing ORF63 but lacking the putative nuclear localization signal of the protein resulted in increased expression of the protein in the cytoplasm, we found that ORF63 protein remained in the nucleus in cells infected with a VZV ORF63 nuclear localization signal deletion mutant. This mutant was not impaired for growth in cell culture or for latency in rodents. Replacement of five serine or threonine phosphorylation sites in ORF63 with alanines resulted in a virus that was impaired for replication in vitro and for latency. A series of ORF63 carboxy-terminal mutants showed that the last 70 amino acids do not affect replication in vitro or latency in rodents; however, the last 108 amino acids are important for replication and latency. Thus, regions of ORF63 that are important for replication in vitro are also required for efficient establishment of latency.

scholarly journals Residues near the Amino Terminus of Rns Are Essential for Positive Autoregulation and DNA Binding

2008 ◽  
Vol 190 (7) ◽  
pp. 2279-2285 ◽  
Author(s):  
Georgeta N. Basturea ◽  
Maria D. Bodero ◽  
Mario E. Moreno ◽  
George P. Munson
Keyword(s):  
Dna Binding ◽  
Amino Terminus ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain ◽  
Regulated Promoters

ABSTRACT Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.

scholarly journals The carboxyl terminus of phage HK022 Nun includes a novel zinc-binding motif and a tryptophan required for transcription termination

2000 ◽  
Vol 14 (6) ◽  
pp. 731-739
Author(s):  
Randolph S. Watnick ◽  
Stephanie Chiyoko Herring ◽  
Arthur G. Palmer ◽  
Max E. Gottesman
Keyword(s):  
Rna Polymerase ◽  
Rna Binding ◽  
Transcription Termination ◽  
Tryptophan Residue ◽  
Binding Motif ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

The amino-terminal arginine-rich motif of the phage HK022 Nun protein binds phage λ nascent mRNA transcripts while the carboxy-terminal domain binds RNA polymerase and arrests transcription. The role of specific residues in the carboxy-terminal domain in transcription termination were investigated by mutagenesis, in vitro and in vivo functional assays, and NMR spectroscopy. Coordination of zinc to three histidine residues in the carboxy-terminus inhibited RNA binding by the amino-terminal domain; however, only two of these histidines were required for transcription arrest. These results suggest that additional zinc-coordinating residues are supplied by RNA polymerase in the context of the Nun–RNA polymerase complex. Substitution of the penultimate carboxy-terminal tryptophan residue with alanine or leucine blocks transcription arrest, whereas a tyrosine substitution is innocuous. Wild-type Nun fails to arrest transcription on single-stranded templates. These results suggest that Nun inhibition of transcription elongation is due in part to interactions between the carboxy-terminal tryptophan of Nun and double-stranded DNA, possibly by intercalation. A model for the termination activity of Nun is developed on the basis of these data.

scholarly journals The Amino-Terminal 100 Residues of the Nitrogen Assimilation Control Protein (NAC) Encode All Known Properties of NAC from Klebsiella aerogenes and Escherichia coli

1999 ◽  
Vol 181 (3) ◽  
pp. 934-940 ◽  
Author(s):  
Wilson B. Muse ◽  
Robert A. Bender
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Nitrogen Assimilation ◽  
Factor Xa ◽  
Klebsiella Aerogenes ◽  
Reading Frame ◽  
Amino Terminal ◽  
Control Protein

ABSTRACT The nitrogen assimilation control protein (NAC) fromKlebsiella aerogenes or Escherichia coli(NACK or NACE, respectively) is a transcriptional regulator that is both necessary and sufficient to activate transcription of the histidine utilization (hut) operon and to repress transcription of the glutamate dehydrogenase (gdh) operon in K. aerogenes. Truncated NAC polypeptides, generated by the introduction of stop codons within thenac open reading frame, were tested for the ability to activate hut and repress gdh in vivo. Most of the NACK and NACE fragments with 100 or more amino acids (wild-type NACK and NACE both have 305 amino acids) were functional in activating hut and repressing gdh expression in vivo. Full-length NACK and NACE were isolated as chimeric proteins with the maltose-binding protein (MBP). NACK and NACE released from such chimeras were able to activatehut transcription in a purified system in vitro, as were NACK129 and NACE100 (a NACKfragment of 129 amino acids and a NACE fragment of 100 amino acids) released from comparable chimeras. A set of NACE and NACK fragments carrying nickel-binding histidine tags (his6) at their C termini were also generated. All such constructs derived from NACE were insoluble, as was NACE itself. Of the his6-tagged constructs derived from NACK, NACK100 was inactive, but NACK120 was active. Several NAC fragments were tested for dimerization. NACK120-his6 and NACK100-his6 were dimers in solution. MBP-NACK and MBP-NACK129 were monomers in solution but dimerized when the MBP was released by cleavage with factor Xa. MBP-NACE was readily cleaved by factor Xa, but the resulting NACE was also degraded by the protease. However, MBP-NACE-his6 was completely resistant to cleavage by factor Xa, suggesting an interaction between the C and N termini of this protein.

scholarly journals Translational and Structural Requirements of the Early Nodulin Gene enod40, a Short-Open Reading Frame-Containing RNA, for Elicitation of a Cell-Specific Growth Response in the Alfalfa Root Cortex

2001 ◽  
Vol 21 (1) ◽  
pp. 354-366 ◽  
Author(s):  
Carolina Sousa ◽  
Christina Johansson ◽  
Celine Charon ◽  
Hamid Manyani ◽  
Christof Sautter ◽  
...  
Keyword(s):  
Amino Acids ◽  
Biological Activity ◽  
Rna Structure ◽  
Open Reading Frames ◽  
In Vitro Translation ◽  
Cortical Cells ◽  
Root Cortex ◽  
Reading Frame ◽  
Early Nodulin

ABSTRACT A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin geneenod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5′ and 3′ regions ofenod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNAs.

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