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E2Fs family includes transcription factors in nucleus of eukaryotic cells. There are eight members of this family from E2F 1 to 8 which activate in G1 and S phases of a cell cycle (Attwooll, 2004). The structure the E2Fs family contains four mainly functional domain a cyclin domain, a DNA binding domain (DBD), dimerization domain, transactivation domain containing Rb binding domain (DeGregori and Johnson, 2006). The DBD of the eight factors are highly homologous to respond to the E2F - DNA interaction, especially E2F 1-5 with a consensus sequence. This consensus sequence, approximately contains 70 residues, is composed of three alpha helices and beta strands (Zheng et al, 1999). In transcription, the first and second helices contribute to bind and stabilise with DPs family through hydrophobic residues especially Leucine residues, while the third helix with a RRIYD motif specifically interacts with a TTTCGCGC motif of DNA (Cress and Nevins, 1996; Johnson et al, 1993; Zheng et al, 1999). Moreover, E2F 1-5 have a nuclear localization signal (NLS), so they are considered as activators in transcriptional activities (Ramirez-Parra et al, 1999). However, E2F 4-5 is occasionally repressors because of absence of the NLC in G0 phase (Falco, 2006). Whereas, E2F 6-8, are probably repressors because of lacks of the NLS and transactivation, especially E2F 7-8 containing only DBDs (Lyon, 2006; Rowland and Bernard, 2006). Besides, E2Fs family are also influenced by DP family and retinoblastoma proteins (pRb), p130, p107 without E2F 7-8 (Chen, 2009; Helin et al, 1992; Wu et al, 1995). Activators are induced through incorporating with DPs to construct a heterodimer complex with a high affinity with targeted genes. Nevertheless, this complex could be repressed by pRbs to become a repressor, especially in G0 phrase (Falco et al, 2006).
The result analysis in the experiment.
In the finger 2A, a transcription of an E2F1 normally acts with the responsive gene through the existence or absence of DP, RP in cell cycle. The E2F interacts with DNA through the DBD in its structure as a dimerization. Due to affected pRb of host cells, the E2F1 probably approaches form 50% to 60% of efficient activation (Wu, 1995; Lukas, 1996). This explains an E2F1 mutant, is unable to bind pRb of host cells, and has the most efficient activation. In another aspect, a Bamdaraâ€™s study (1993) showed that incorporation between E2F1 and DP1 creates a heterodimer complex improving interacting DNA of the E2F1 when individual DPs could not interact with DNA (Wu et al, 1995). Besides, the E2F1 mutant, which could be not bound by pRB, has the most efficiency in the experiment (Lee, 2002). Hence, the transcription of E2F is less efficient than the E2F1-DP complex and the control positive experiments.
On the other hand, in finger 2B, there is probably a problem in the PCR products of the new E2F1 that could lead to inactivate its transcription in this experiment. In fact, results of the new E2F1 are similarly in experiment with present and absence of DP and pRb, whereas the positive control of the E2F1 mutant has a high expression. This phenomenon means that the new E2F1 do not interact with plasmid of luciferase.
The ORF finder program is used to translate the cDNA of the new E2F1 becoming the protein sequence alignment. This alignment is then compared with the sequence alignment of the E2F1. According to this comparison, two residues which are Leucine (Leu) and Asparagine (Asn) residues of the sequence of the E2F1 are replaced by Glutamic acid (Glu) and Phenyalanin (Phe) residues in the new E2F1 respectively. Moreover, these residues are in the DNA binding site of the E2F1 which plays a significant role in DNA binding, especially Leu residue being a high conversed residue of the E2Fs family. Therefore, we could infer that the new E2F1 can not bind to E2F- responsive promoters of the luciferase reporter plasmid.
The ORF finder program is used to find down protein alignment of the new E2F1. the two residues (red box ) in consensus sequence (yellow box), are Leu and Asn residues, are changed by Glu and Phe residues in the consevered region of E2F1 (yellow box) Unfortunatelly, this DNA binding domain (DBD) of the E2Fs family, is highly conserved in structure of the E2Fs family, plays a significant role in binding E2F1-responsive promoters (Croxton, 2002). Futhermore, several studies showed that structural modifications of this domain could lead to the inactivation of E2F1 (Wu et al, 1995; Hsieh, 1997). In addition, an E2F8 study of Maiti and his colleagues (2005) indicated that a change of the conversed Leu residue could result in an inactivated transcription of E2F1. This result might be explained due to specific position and the hydrophobic properties of the Leu residue.
In the E2F1, the Leu residue, is in the first helix of DBD, is a highly conserved and hydrophobic residue, plays a significant role in interacting with DNA and the DP. A mutation which appears at the conserved Leu residue of a first helix in the DBD likely results in unbind to DNA through the DBD of the new E2F1 (Cress, 1993; Johnson, 1993). Furthermore, the Leu residue also contributes to an interaction between DP and E2F1 through hydrophobic surfaces o their DBD to create a heterodimer complex (Zheng et al, 1999). This E2F-DP complex is probably high efficient to bind to DNA through a homology in their DBDs (Girling et al, 1993, Zheng et al, 1999). In fact, the function of DBD studies argue that two DBDs in a protein could be effectively impacted on DNA binding (Kosugi and Ohashi, 2002; Logan et al, 2004). Therefore, the conserved Leu residue contributes to improve the transcription capacity of the E2F1 - DP complex. As a result of lack of the conserved Leu residue, the new E2F1 could not bind E2F responsive promoter of luciferase genes and inactivate a transcription of the new E2F1.
To conform this hypothesis that the new E2F1 construct did not bind both DP and pRB, their monoclonal antibodies could be use to determine the presence of these E2F1-DP pRb complexes in cell extracts in these experiments (Wu, 1995). Unless the complexes of the new E2F1 with either DP or pRB existed in these soluble extracts, E2F1 antibodies would be individually conformed without DP or pRb antibodies in these complexes. Therefore, the role of residue mutants could be indicated in the E2F1 DNA interaction through above results. This method only assists in finding the effects of an expected mutant in DBD. However, if the mutant is a technically error, the best solution will be fixing optimal demand in PRC and cloning pathways (Silver and Hunt, 1993).
The sequence of the new E2F1 is created by the ORF finder: MALAGAPAGGPCAPALEALLGAGALRLLDSSQIVIISAAQDASAPPAPTGPAAPAAGPCDPDLLLFATPQAPRPTPSAPRPALGRPPVKRRLDLETDHQYLAESSGPARGRGRHPGKGVKSPGEKSRYETSEFLTTKRFLELLSHSADGVVDLNWAAEVLKVQKRRIYDITNVLEGIQLIAKKSKNHIQWLGSHTTVGVGGRLEGLTQDLRQLQESEQQLDHLMNICTTQLRLLSEDTDSQRLAYVTCQDLRSIADPAEQMVMVIKAPPETQLQAVDSSENFQISLKSKQGPIDVFLCPEETVGGISPGKTPSQEVTSEEENRATDSATIVSPPPSSPPSSLTTDPSQSLLSLEQEPLLSRMGSLRAPVDEDRLSPLVAADSLLEHVREDFSGLLPEEFISLSPPHEALDYHFGLEEGEGIRDLFDCDFGDLTPLDF
The sequence of the native E2F-1: