Faculty Opinions recommendation of A comprehensive alanine scanning mutagenesis of the Escherichia coli transcriptional activator SoxS: identifying amino acids important for DNA binding and transcription activation.

2003 ◽  
Author(s):  
Juan Luis Ramos
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Dna Binding ◽  
Transcriptional Activator ◽  
Transcription Activation ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning

scholarly journals Residues Required for Bacillus subtilis PhoP DNA Binding or RNA Polymerase Interaction: Alanine Scanning of PhoP Effector Domain Transactivation Loop and α Helix 3

2004 ◽  
Vol 186 (5) ◽  
pp. 1493-1502 ◽  
Author(s):  
Yinghua Chen ◽  
Wael R. Abdel-Fattah ◽  
F. Marion Hulett
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Transcription Activation ◽  
Phosphate Deficiency ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Α Helix

ABSTRACT Bacillus subtilis PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independent of its phosphorylation state, phosphorylation of PhoP may optimize DNA binding or the interaction with RNA polymerase. We describe alanine scanning mutagenesis of the PhoP α loop and α helix 3 region of PhoPC (Val190 to E214) and functional analysis of the mutated proteins. Eight residues important for DNA binding were clustered between Val202 and Arg210. Using in vivo and in vitro functional analyses, we identified three classes of mutated proteins. Class I proteins (PhoPI206A, PhoPR210A, PhoPL209A, and PhoPH208A) were phosphorylation proficient and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Class II proteins (PhoPH205A and PhoPV204A) were phosphorylation proficient and could dimerize but could not bind DNA prior to phosphorylation. Members of this class had higher transcription activation in vitro than in vivo. The class III mutants, PhoPV202A and PhoPD203A, had a reduced rate of phosphotransfer and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Seven alanine substitutions in PhoP (PhoPV190A, PhoPW191A, PhoPY193A, PhoPF195A, PhoPG197A, PhoPT199A, and PhoPR200A) that specifically affected transcription activation were broadly distributed throughout the transactivation loop extending from Val190 to as far toward the C terminus as Arg200. PhoPW191A and PhoPR200A could not activate transcription, while the other five mutant proteins showed decreased transcription activation in vivo or in vitro or both. The mutagenesis studies may indicate that PhoP has a long transactivation loop and a short α helix 3, more similar to OmpR than to PhoB of Escherichia coli.

scholarly journals Comprehensive Alanine-scanning Mutagenesis of Escherichia coli CsrA Defines Two Subdomains of Critical Functional Importance

2006 ◽  
Vol 281 (42) ◽  
pp. 31832-31842
Author(s):  
Jeffrey Mercante ◽  
Kazushi Suzuki ◽  
Xiaodong Cheng ◽  
Paul Babitzke ◽  
Tony Romeo
Keyword(s):  
Escherichia Coli ◽  
Functional Importance ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning

scholarly journals Two Domains Unique to Osteoblast-Specific Transcription Factor Osf2/Cbfa1 Contribute to Its Transactivation Function and Its Inability To Heterodimerize with Cbfβ

1998 ◽  
Vol 18 (7) ◽  
pp. 4197-4208 ◽  
Author(s):  
Kannan Thirunavukkarasu ◽  
Muktar Mahajan ◽  
Keith W. McLarren ◽  
Stefano Stifani ◽  
Gerard Karsenty
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Critical Role ◽  
Transcription Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Amino Terminal ◽  
Related Proteins ◽  
Repression Domain

ABSTRACT Osf2/Cbfa1, hereafter called Osf2, is a member of the Runt-related family of transcription factors that plays a critical role during osteoblast differentiation. Like all Runt-related proteins, it contains a runt domain, which is the DNA-binding domain, and a C-terminal proline-serine-threonine-rich (PST) domain thought to be the transcription activation domain. Additionally, Osf2 has two amino-terminal domains distinct from any other Runt-related protein. To understand the mechanisms of osteoblast gene regulation by Osf2, we performed an extensive structure-function analysis. After defining a short Myc-related nuclear localization signal, a deletion analysis revealed the existence of three transcription activation domains and one repression domain. AD1 (for activation domain 1) comprises the first 19 amino acids of the molecule, which form the first domain unique to Osf2, AD2 is formed by the glutamine-alanine (QA) domain, the second domain unique to Osf2, and AD3 is located in the N-terminal half of the PST domain and also contains sequences unique to Osf2. The transcription repression domain comprises the C-terminal 154 amino acids of Osf2. DNA-binding, domain-swapping, and protein interaction experiments demonstrated that full-length Osf2 does not interact with Cbfβ, a known partner of Runt-related proteins, whereas a deletion mutant of Osf2 containing only the runt and PST domains does. The QA domain appears to be responsible for preventing this heterodimerization. Thus, our results uncover the unique functional organization of Osf2 by identifying functional domains not shared with other Runt-related proteins that largely control its transactivation and heterodimerization abilities.

scholarly journals Hydrophilic Domain II of Escherichia coli Dr Fimbriae Facilitates Cell Invasion

2005 ◽  
Vol 73 (9) ◽  
pp. 6119-6126 ◽  
Author(s):  
Margaret Das ◽  
Audrey Hart-Van Tassell ◽  
Petri T. Urvil ◽  
Susan Lea ◽  
David Pettigrew ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Cell Invasion ◽  
Cell Entry ◽  
Wild Type ◽  
Alanine Scanning Mutagenesis ◽  
Decay Accelerating Factor ◽  
Fimbrial Subunit ◽  
Hydrophilic Domain ◽  
Intracellular Compartmentalization

ABSTRACT Uropathogenic and diarrheal Escherichia coli strains expressing adhesins of the Dr family bind to decay-accelerating factor, invade epithelial cells, preferentially infect children and pregnant women, and may be associated with chronic or recurrent infections. Thus far, no fimbrial domain(s) that facilitates cell invasion has been identified. We used alanine scanning mutagenesis to replace selected amino acids in hydrophilic domain II of the structural fimbrial subunit DraE and evaluated recombinant mutant DraE for attachment, invasion, and intracellular compartmentalization. The mutation of amino acids V28, T31, G33, Q34, T36, and P40 of DraE reduced or abolished HeLa cell invasion but did not affect attachment. Electron micrographs showed a stepwise entry and fusion of vacuoles containing Escherichia coli mutants T36A and Q34A or corresponding beads with lysosomes, whereas vacuoles with wild-type Dr adhesin showed no fusion. Mutants T31A and Q34A, which were deficient in invasion, appeared to display a reduced capacity for clustering decay-accelerating factor. Our findings suggest that hydrophilic domain II may be involved in cell entry. These data are consistent with the interpretation that in HeLa cells the binding and invasion phenotypes of Dr fimbriae may be separated.

Identification of functional regions in the yeast transcriptional activator ADR1

1988 ◽  
Vol 8 (5) ◽  
pp. 2125-2131
Author(s):  
L T Bemis ◽  
C L Denis
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Glucose Repression ◽  
Transcriptional Activator ◽  
Dna Binding Protein ◽  
Functional Regions ◽  
Zinc Finger Motifs ◽  
Repressor Molecule ◽  
Carboxy Terminal

The transcriptional activator ADR1 from Saccharomyces cerevisiae is a postulated DNA-binding protein that controls the expression of the glucose-repressible alcohol dehydrogenase (ADH2). Carboxy-terminal deletions of the ADR1 protein (1,323 amino acids in length) were used to localize its functional regions. The transcriptional activation region was localized to the N-terminal 220 amino acids of ADR1 containing two DNA-binding zinc finger motifs. In addition to the N terminus, a large part of the ADR1 sequence was shown to be essential for complete activation of ADH2. Deletion of the putative phosphorylation region, defined by ADR1c mutations that overcome glucose repression, did not render ADH2 expression insensitive to glucose repression. Instead, this region (amino acids 220 through 253) was found to be required by ADR1 to bypass glucose repression. These results suggest that ADR1c mutations enhance ADR1 function, rather than block an interaction of the putative phosphorylation region with a repressor molecule. Furthermore, the protein kinase CCR1 was shown to affect ADH2 expression when the putative phosphorylation region was removed, indicating that CCR1 does not act solely through this region. A functional ADR1 gene was also found to be necessary for growth on glycerol-containing medium. The N-terminal 506 amino acids of ADR1 were required for this newly identified function, indicating that ADH2 activation and glycerol growth are controlled by separate regions of ADR1.

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