scholarly journals RNA Polymerase Tags To Monitor Multidimensional Protein–Protein Interactions Reveal Pharmacological Engagement of Bcl-2 Proteins

2017 ◽  
Vol 139 (34) ◽  
pp. 11964-11972 ◽  
Author(s):  
Jinyue Pu ◽  
Jeffrey A. Dewey ◽  
Abbas Hadji ◽  
James L. LaBelle ◽  
Bryan C. Dickinson
Keyword(s):  
Rna Polymerase ◽  
Protein Interactions ◽  
Protein Protein Interactions

scholarly journals Interaction between Human Respiratory Syncytial Virus (RSV) M2-1 and P Proteins Is Required for Reconstitution of M2-1-Dependent RSV Minigenome Activity

2003 ◽  
Vol 77 (19) ◽  
pp. 10670-10676 ◽  
Author(s):  
Stephen W. Mason ◽  
Erika Aberg ◽  
Carol Lawetz ◽  
Rachel DeLong ◽  
Paul Whitehead ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Rna Polymerase ◽  
Protein Interactions ◽  
Luciferase Reporter ◽  
Protein Protein Interactions ◽  
Luciferase Reporter Gene ◽  
Alanine Scanning Mutagenesis ◽  
Syncytial Virus ◽  
P Proteins ◽  
Rna Polymerase Subunits

ABSTRACT We have investigated protein-protein interactions among the respiratory syncytial virus (RSV) RNA polymerase subunits using affinity chromatography. Here we demonstrate a novel interaction of P and M2-1 proteins. Phosphorylation of either M2-1 or P appears to be dispensable for this interaction. Internal deletions within P mapped the M2-1-binding domain to a region between residues 100 and 120. Alanine-scanning mutagenesis within this region of P revealed that substitution of any one of the three residues, L101, Y102, and F109, prevented both M2-1 and P binding and expression of an M2-1-dependent luciferase reporter gene. However, these same mutations did not prevent the activity of an M2-1-independent chloramphenicol acetyltransferase minigenome, suggesting that these residues of P specifically affect M2-1-P interaction. On the basis of these observations, it is possible that the interaction between RSV M2-1 and P proteins is important for viral replication.

scholarly journals In culture cross-linking of bacterial cells reveals proteome scale dynamic protein-protein interactions at the peptide level

2016 ◽  
Author(s):  
Luitzen de Jong ◽  
Edward A. de Koning ◽  
Winfried Roseboom ◽  
Hansuk Buncherd ◽  
Martin J. Wanner ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Protein Interactions ◽  
Cross Linking ◽  
Bacterial Cells ◽  
Protein Protein Interactions ◽  
Genome Wide ◽  
Peptide Level ◽  
Complex Protein ◽  
Genome Wide Data ◽  
Cross Links

AbstractIdentification of dynamic protein-protein interactions at the peptide level on a proteomic scale is a challenging approach that is still in its infancy. We have developed a system to cross-link cells directly in culture with the special lysine cross-linker bis(succinimidyl)-3-azidomethyl-glutarate (BAMG). We used the Gram positive model bacteriumBacillus subtilisas an exemplar system. Within 5 min extensive intracellular cross-linking was detected, while intracellular cross-linking in a Gram-negative species,Escherichia coli, was still undetectable after 30 min, in agreement with the low permeability in this organism for lipophilic compounds like BAMG. We were able to identify 82 unique inter-protein cross-linked peptides with less than a 1% false discovery rate by mass spectrometry and genome-wide data base searching. Nearly 60% of the inter-protein cross-links occur in assemblies involved in transcription and translation. Several of these interactions are new, and we identified a binding site between the δ and β′ subunit of RNA polymerase close to the downstream DNA channel, providing a clue into how δ might regulate promoter selectivity and promote RNA polymerase recycling. Our methodology opens new avenues to investigate the functional dynamic organization of complex protein assemblies involved in bacterial growth.

scholarly journals Evidence that Negative Elongation Factor Represses Transcription Elongation through Binding to a DRB Sensitivity-Inducing Factor/RNA Polymerase II Complex and RNA

2002 ◽  
Vol 22 (9) ◽  
pp. 2918-2927 ◽  
Author(s):  
Yuki Yamaguchi ◽  
Naoto Inukai ◽  
Takashi Narita ◽  
Tadashi Wada ◽  
Hiroshi Handa
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Protein Interactions ◽  
Rna Binding ◽  
Elongation Factor ◽  
Protein Protein Interactions ◽  
Negative Elongation Factor ◽  
Nascent Transcripts

ABSTRACT Negative elongation factor (NELF) is a human transcription factor complex that cooperates with DRB sensitivity-inducing factor (DSIF)/hSpt4-hSpt5 to repress elongation by RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, including NELF-A, a candidate gene product for Wolf-Hirschhorn syndrome, and NELF-E, a putative RNA-binding protein with arginine-aspartic acid (RD) dipeptide repeats. Here we report several important findings regarding the DSIF/NELF-dependent elongation control. First, we have established an effective method for purifying the active NELF complex using an epitope-tagging technique. Second, the five polypeptides each are important and together are sufficient for its function in vitro. Third, NELF does not bind to either DSIF or RNAPII alone but does bind to the preformed DSIF/RNAPII complex. Fourth, NELF-E has a functional RNA-binding domain, whose mutations impair transcription repression without affecting known protein-protein interactions. Taken together, we propose that NELF causes RNAPII pausing through binding to the DSIF/RNAPII complex and to nascent transcripts. These results also have implications for how DSIF and NELF are regulated in a gene-specific manner in vivo.

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