Binding of site-directed monoclonal antibodies to an epitope located in the A/B region (amino acids 140–154) of human estrogen receptor-induced conformational changes in an epitope in the DNA-binding domain

Steroids ◽  
1996 ◽  
Vol 61 (9) ◽  
pp. 549-556 ◽  
Author(s):  
Abdulmaged M. Traish ◽  
Matthew Pavao
1999 ◽  
Vol 73 (5) ◽  
pp. 4475-4480 ◽  
Author(s):  
Tetsuya Ishikawa ◽  
Nobuo Okui ◽  
Noriko Kobayashi ◽  
Ryuta Sakuma ◽  
Tadaichi Kitamura ◽  
...  

ABSTRACT Integrase of human immunodeficiency virus type 1 (HIVIN) consists of 288 amino acids, and its minimum DNA-binding domain (MDBD) (amino acids [aa] 220 to 270) is required for the integration reaction. We produced and characterized four murine monoclonal antibodies (MAbs) to the MDBD of HIVIN (strain LAI). Immunoblot and enzyme-linked immunosorbent assays with truncated HIVINs showed that those MAbs recognized sequential epitopes within the MDBD (aa 228 to 236, 237 to 252, 253 to 261, and 262 to 270). Their binding to HIVIN inhibited terminal cleavage and strand transfer activities but not disintegration activity in vitro. This collection of MAbs is useful for studying the structure and function of the MDBD by complementing mutational analyses and other biochemical studies.


1990 ◽  
Vol 10 (10) ◽  
pp. 5128-5137 ◽  
Author(s):  
M M Witte ◽  
R C Dickson

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.


1999 ◽  
Vol 19 (4) ◽  
pp. 2880-2886 ◽  
Author(s):  
Asish K. Ghosh ◽  
Robert Steele ◽  
Ratna B. Ray

ABSTRACT We initially identified c-myc promoter binding protein 1 (MBP-1), which negatively regulates c-myc promoter activity, from a human cervical carcinoma cell expression library. Subsequent studies on the biological role of MBP-1 demonstrated induction of cell death in fibroblasts and loss of anchorage-independent growth, reduced invasive ability, and tumorigenicity of human breast carcinoma cells. To investigate the potential role of MBP-1 as a transcriptional regulator, a chimeric protein containing MBP-1 fused to the DNA binding domain of the yeast transactivator factor GAL4 was constructed. This fusion protein exhibited repressor activity on the herpes simplex virus thymidine kinase promoter via upstream GAL4 DNA binding sites. Structure-function analysis of mutant MBP-1 in the context of the GAL4 DNA binding domain revealed that MBP-1 transcriptional repressor domains are located in the N terminus (amino acids 1 to 47) and C terminus (amino acids 232 to 338), whereas the activation domain lies in the middle (amino acids 140 to 244). The N-terminal domain exhibited stronger transcriptional repressor activity than the C-terminal region. When the N-terminal repressor domain was transferred to a potent activator, transcription was strongly inhibited. Both of the repressor domains contained hydrophobic regions and had an LXVXL motif in common. Site-directed mutagenesis in the repressor domains indicated that the leucine residues in the LXVXL motif are required for transcriptional repression. Mutation of the leucine residues in the common motif of MBP-1 also abrogated the repressor activity on the c-mycpromoter. In addition, the leucine mutant forms of MBP-1 failed to suppress cell growth in fibroblasts like wild-type MBP-1. Taken together, our results indicate that MBP-1 is a complex cellular factor containing multiple transcriptional regulatory domains that play an important role in cell growth regulation.


1993 ◽  
Vol 13 (12) ◽  
pp. 7496-7506
Author(s):  
X Mao ◽  
M K Darby

Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.


1996 ◽  
Vol 271 (33) ◽  
pp. 20053-20059 ◽  
Author(s):  
Kirk Ekena ◽  
Karen E. Weis ◽  
John A. Katzenellenbogen ◽  
Benita S. Katzenellenbogen

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