Separate DNA binding and transcription activation domains
(a)DNA binding domains. Following are some of the protein motifs, which are involved in regulation of transcription through DNA binding : (i) several steroid receptors are activated by corresponding steroids, leading to binding of these receptors to DNA thus initiating transcription; (ii) zinc finger motif has a DNA binding domain, which was recognized for the first time in transcription factor TFHIA, required for transcription of 5S rRNA by RNA polymerase III (see later for details);
(i) Steroid receptors. The steroid receptors include receptors for the steroid hormones, retinoids, vitamin D, thyroid hormones and a number of other compounds. These proteins contain separate domains for hormone binding, DNA binding and for transcriptional activation. The DNA binding domain contains 70 amino acid residues, with eight conserved cysteine residues, forming two zinc fingers with zinc. A peptide from this domain, can fold (into two a helices) in the presence of zinc, and is utilized for recognizing the appropriate binding site in DNA.
Cys2/His2 fingers have the following consensus sequence : Cys—X2—4—Cys—X3—Phe—X5—Leu-X2-His-X3-His. Each finger consists of about 23 amino acids, linked to another finger by 7-8 amino acids. Details of some transcription factors with zinc fingers are listed in Table 32.4. These zinc fingers are required for binding to DNA. On one extreme, these fingers may involve almost the entire protein as in TFIIIA (9 fingers), and on the other extreme only a small domain is involved in forming zinc fingers, as in ADR1 (2 fingers). Since zinc fingers are found in several known transcription factors, this feature also helped in recognizing proteins that may function as transcription factors, e.g. TDF (testis determining factor), ZFX, ZFY (zinc finger proteins encoded by genes on human X and Y chromosomes).
Translation initiation factors (e.g. eIF2B) have also been shown to have zinc fingers which help in recognition of initiation codons.
Among eukaryotic regulatory proteins, the most important domain with HTH motif is the 60-residues long homeodomain motif found in the products of a number of developmental genes in Drosophila. The polypeptide chain representing homeodomain, folds as a 3-helix bundle, in which second and third helices form the HTH.
Fig. 32.21. Association of leucine zippers : (a) antiparallel zippering (as earlier envisaged); (b) parallel zippering due to side-by-side overlapping.
Fig. 32.22. A consensus sequence in protruding arms of leucine zippers, based on a comparison of several proteins; regions that aid in binding and touch the DNA are highlighted
Fig. 32.21. Association of leucine zippers : (a) antiparallel zippering (as earlier envisaged); (b) parallel zippering due to side-by-side overlapping.
Fig. 32.22. A consensus sequence in protruding arms of leucine zippers, based on a comparison of several proteins; regions that aid in binding and touch the DNA are highlighted
Fig. 32.23. DNA binding regions of leucine zipper : (a) alpha helices protruding out from DNA (as earlier envisaged); (b) the. protruding regions of alpha helices bend at asparagine residues forming a Y-shaped dimer, to establish a grip on DNA motif.
Fig. 32.24. Three types of protein domains responsible for transcriptional activation by DNA binding factors.
Fig. 32.25. Mechanism of action of activation domains of transcription factors : (a) a hypothetical array of cis elements in the promoter/enhancer regions of a gene transcribed by Pol II, and the associated transcription factors (all these DNA binding factors may not be required simultaneously to initiate transcription); (b) mechanism(s) by which cis elements activate transcription may involve protein-protein interaction, so that distally found factors can take part in transcription initiation.
Fig. 32.26. Similarity of several regions of E. coli sigma factor (σ70), with those in three nuclear transcription factors, i.e. RPO24 (a subunit of yeast RNA polymerase II), RAP30 (a human transcription factor) and TFIID; and one mitochondrial transcription factor, MTF1.
Fig. 32.23. DNA binding regions of leucine zipper : (a) alpha helices protruding out from DNA (as earlier envisaged); (b) the. protruding regions of alpha helices bend at asparagine residues forming a Y-shaped dimer, to establish a grip on DNA motif.
(b) Transcription activation domains. The transcription activation domains are separate from DNA binding domains and each consists of 30-100 amino acids. Different types of activation domains have been exchanged and combined with other DNA binding proteins to produce chimeric transcription factors. The transcription activation domains function through protein-protein interactions and often help in establishing contacts with components of transcription complex, which leads to activation of transcription. Following three types of activation domains, shown in Figure 32.24, have been identified : (i) Acidic domains were first identified in yeast transcription factors GAL4 and GCN4. Later they were found in glucocorticoid hormone receptor and also in AP-1/Jun transcription factors. They have two features in common, i.e. there are regions of significant negative charge and they can form amphipathic alpha helical structures. The acidic domains help in association of TFIIB and TFIID. (ii) Glutamine rich domains were tirst identified in SP1, and later also in Drosophila's antennapaedia and ultrabithorax, in yeast-'s HAP1, HAP2, and GAL11 and in several mammalian factors (OCT-1, OCT-2, JUN, AP-2, SRF). (iii) Proline rich domains havebeen identified in CTF/NF-1 and in several other mammalian factors (AP-2, JUN, OCT-2, SRF). The mechanisms of action of these activation domains are shown in Figure 32.25.
Fig. 32.24. Three types of protein domains responsible for transcriptional activation by DNA binding factors.
Fig. 32.25. Mechanism of action of activation domains of transcription factors : (a) a hypothetical array of cis elements in the promoter/enhancer regions of a gene transcribed by Pol II, and the associated transcription factors (all these DNA binding factors may not be required simultaneously to initiate transcription); (b) mechanism(s) by which cis elements activate transcription may involve protein-protein interaction, so that distally found factors can take part in transcription initiation.
Proteins that influence transcription without binding to DNA. Several proteins, which influence transcription without binding to DNA (sometimes through recognition of another protein) include the following : (i) E1A activates Ad (adenovirus) genes, pol II and pol HI genes, (ii) Tat acts at post-transcriptional level with the help of tar sequence in HIV RNA and (iii) Vmw 65 activates initial early (IE) genes in HSV (herpes simplex virus), by associating with another transcription factor.
Similarity between prokaryotic sigma factor and some eukaryotic transcription factors. Recently, similarity between sigma factor and atleast three different nuclear mRNA transcription factors has been demonstrated. These transcription factors include the following : (i) RPO24, which is the fourth . largest subunit of yeast RNA polymerase Il.(ii) RAP30, which is a part of a co'mplex of two proteins that bind tightly to human RNA polymerase II. (iii) TFIID, which binds to TATA box. The similarities of certain domains of sigma with these three factors are shown in Figure 32.26.
The above similarities are consistent with the observation that the two largest subunits of eukaryotic nuplear RNA polymerases are homologues of the large subunits of bacterial RNA polymerase.