Question

please help me with these questions:

1. Describe the DNA-binding domains of four major families of transcription factors and their interaction with DNA.
2. Describe how CTD phosphorylation affects transcription.

Thanks

Answer 1

(1) SOLUTION :

Transcription factors are modular proteins composed of distinct and seperable functional domains:

• DNA binding domain (DBD) interacts with specific DNA sequences
• Transcription Activation domain (TAD) interacts with other proteins (general transcription machinery, coactivators etc., to stimulate transcription from the core promoter

Transcription factors that bind DNA do so in a sequence specific manner.

The three common features most DNA binding proteins are:

• They bind to the major groove (12Å wide and 8Å deep ) of B- DNA through α –helices.
• The minor groove of B-DNA (5Å wide, 8Å deep) is generally too narrow to fit entire α –helices.
• They generally do not disrupt the base pairs of the DNA, but distort the conformation of the backbone by bending the double helix

DNA binding domains of eukaryotic transcription factors

In general, transcription factors which bind DNA in a sequence-specific manner contain characteristic DNA binding domains or motifs.Infact, eukaryotic transcription factors are categorized into distinct families based on the type of DNA binding motifs they contains the following

Eukaryotic transcription factor families

1. Helix-Turn-Helix proteins
2. Zinc finger proteins
3. Leucine zipper protein
4. Helix-Loop-Helix proteins

1) Helix-turn-Motif

Helix-turn-helix motif of protein Amino acid sequence forms a turn a-helix a-helix

The helix-turn-helix motif was initially identified as the DNA-binding domain of bacteriophage repressors.These repressors contain two alpha helices: one that lies in the major groove of DNA and the other lies at an angle across DNA.Two adjacent α-helices separated by a "turn" of several amino acids enables the protein to bind to DNA . However, the HTH motif cannot fold or function alone, but is always part of a larger DNA-binding domain and amino acid residues outside the HTH motif are important in regulating DNA recognition and binding. A motif similar to helix-turn-helix motif is present in certain eukaryotic transcription factors that play a key role in regulation of embryonic development.The DNA binding domain in these proteins is called the homeodomain and the C-terminal region of homeodomain shows homology with the helix-turn-helix motif of procaryotic repressors. it contains 60 amino acid motif known as homeobox.

Fig1.The λ repressor of bacteriophage lambda employs two helix-turn-helix motifs (left; green) to bind DNA (right; blue and red). The λ repressor protein in this image is a dimer

2) zinc finger motif

Histidine ZINC ATOM Cysteine Histidine Cysteine

Certain eukaryotic transcription factors contain a unique DNA binding motif called a zinc-finger where a zinc ion is coordinated by 2 Cysteine and 2 Histidine residues.This motif was first discovered in a transcription factor known as TFIIIA in the frog, Xenopus laevis.The number of zinc fingers vary in different transcription factors.The non-conserved aminoacids in the C-terminal side of each finger recognize specific target sites.The C-terminal part of each finger forms a-helices that bind DNA while the N-terminal part form b-sheets.A C2H2 zinc finger protein has a series of zinc fingers and the consensus sequence of a single finger is:

Cys-X2-4-Cys-X3-Phe-X3-Leu-X2-His-X3-His

The interspersed cysteine and histidine residues covalently bind zinc atoms, folding the amino acids into loops known as zinc fingers.Each finger consists of approximately 23 amino acids (with a loop of 12 to 14 amino acids between the Cys and His residues) and a linker between loops consisting of 7 or 8 amino acids. The amino acids in the loop bind to specific DNA sequences.

MSNLPPTFGSTRQSPEDQSPPVPKELSFNGTTPSGKLRLFVCQTCTRAFARQEHLKRHER 60

SHTKEKPFSCGICSRKFSRRDLLLRHAQKLHSNCSDAAITRLRRKATRRSSNAAGSISGS 120

WI- OLYAMO To zn s - 0 av RL EV KE KP F Sncs

a LYO Ini Idrero TO IZ FO> WO R L E TKE KPFSP S ONDOTI I FO > RTH NE KPF

Zinc-finger proteins, represent one of the major families of eukaryotic transcription factors including those belonging to the steroid hormone receptor super family.The steroid hormone receptors contain C2C2 zinc fingers instead of C2H2 fingers. They have the consensus sequence:

Cys-X2-Cys-X13-Cys-X2-Cys

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In the case of steroid hormone receptors, the first zinc finger determines specificity of DNA binding and the second finger specifies dimerization specificity.It has been shown that a single amino acid change in the base of the first finger can alter the DNA binding specificity of steroid hormone receptors.

3) Basic leucine zipper (bzip)

Leucine zipper Basic region

In certain transcription factors the dimerization domain forms motif known as leucine zipper.This motif consists of two amphipathic helices, one from each subunit, interacting with each other resulting in a left handed coiled-coil structure.The leucine zipper is a interdigitation of regularly spaced leucine residues in one helix with leucines from the adjacent helix.Leucine zipper motifs can mediate homodimer or heterodimer formation.An important point to note is that the leucine zipper motif itself does not play a directly participate in DNA binding.The DNA-binding domain is adjacent to the leucine zipper and the latter facilitates protein-protein dimerization. Dimerization allows the juxtaposition of the DNA-binding regions of each subunit. A leucine zipper forms an amphipathic helix in which the leucines of the on one protein protrude from the alpha helix and interdigitate with the leucines of the other DNA protein in parallel to form a coiled coil domain.The region adjacent to the leucine repeats is highly basic and forms the DNA-binding site.

4) helix-loop-helix (HLH)

C-terminus bo Helix 2 Helix 1 Loop C-terminus N-terminus N-terminus

The amphipathic helix-loop-helix (HLH) motif was identified in certain transcription factors involved in developmental regulation. They contain a stretch of 40-50 aminoacids comprising of two amphipathic a-helices separated by a linker region (the loop) of varying length.The HLH proteins form both homodimers and heterodimers by means of interactions between the hydrophobic residues on the corresponding faces of the 2 helices. The ability to form dimers resides with these amphipathic helices.Most HLH proteins contain a basic region adjacent to the HLH motif that is involved in DNA binding and hence they are often called bHLH proteins.Examples of HLH proteins include MyoD, Myf5, myogenin and MRF4 which are involved in myogenesis and are called myogenic regulatory factors (MRFs)

(2) SOLUTION :

1) C-terminal domain (CTD)

RNA POLII 5. pre-mRNA CARBOXYL TAIL DOMAIN

Fig : RNA Pol II in action, showing the CTD extension to the C-terminal of POLR2A.

The carboxy-terminal domain (CTD) of RNA polymerase II is that portion of the polymerase that is involved in the initiation of DNA transcription, the capping of the RNA transcript, and attachment to the spliceosome for RNA splicing. The CTD typically consists of up to 52 repeats (in humans) of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser.The carboxy-terminal repeat domain (CTD) is essential for life. Cells containing only RNAPII with none or only up to one-third of its repeats are inviable.The CTD is an extension appended to the C terminus of RPB1, the largest subunit of RNA polymerase II. It serves as a flexible binding scaffold for numerous nuclear factors, determined by the phosphorylation patterns on the CTD repeats. Each repeat contains an evolutionary conserved and repeated heptapeptide, Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, which is subjected to reversible phosphorylations during each transcription cycle.This domain is inherently unstructured yet evolutionarily conserved, and in eukaryotes it comprises from 25 to 52 tandem copies of the consensus repeat heptad.As the CTD is frequently not required for general transcription factor (GTF)-mediated initiation and RNA synthesis, it does not form a part of the catalytic essence of RNAPII, but performs other functions.

2) CTD phosphorylation

RNAPII can exist in two forms: RNAPII0, with a highly phosphorylated CTD, and RNAPIIA, with a nonphosphorylated CTD.Phosphorylation occurs principally on Ser2 and Ser5 of the repeats, although these positions are not equivalent. The phosphorylation state changes as RNAPII progresses through the transcription cycle: The initiating RNAPII is form IIA, and the elongating enzyme is form II0. While RNAPII0 does consist of RNAPs with hyperphosphorylated CTDs, the pattern of phosphorylation on individual CTDs can vary due to differential phosphorylation of Ser2 versus Ser5 residues and/or to differential phosphorylation of repeats along the length of the CTD.The PCTD (phosphoCTD of an RNAPII0) physically links pre-mRNA processing to transcription by tethering processing factors to elongating RNAPII, e.g., 5′-end capping, 3′-end cleavage, and polyadenylation.Ser5 phosphorylation (Ser5PO4) near the 5′ ends of genes depends principally on the kinase activity of TFIIH (Kin28 in yeast; CDK7 in metazoans).The transcription factor TFIIH is a kinase and will hyperphosphorylate the CTD of RNAP, and in doing so, causes the RNAP complex to move away from the initiation site. Subsequent to the action of TFIIH kinase, Ser2 residues are phosphorylated by CTDK-I in yeast (CDK9 kinase in metazoans). Ctk1 (CDK9) acts in complement to phosphorylation of serine 5 and is, thus, seen in middle to late elongation.CDK8 and cyclin C (CCNC) are components of the RNA polymerase II holoenzyme that phosphorylate the carboxy-terminal domain (CTD). CDK8 regulates transcription by targeting the CDK7/cyclin H subunits of the general transcription initiation factor IIH (TFIIH), thereby providing a link between the mediator and the basal transcription machinery.The gene CTDP1 encodes a phosphatase that interacts with the carboxy-terminus of transcription initiation factor TFIIF, a transcription factor that regulates elongation as well as initiation by RNA polymerase II.Also involved in the phosphorylation and regulation of the RPB1 CTD is cyclin T1 (CCNT1).Cyclin T1 tightly associates and forms a complex with CDK9 kinase, both of which are involved in the phosphorylation and regulation.

ATP + [DNA-directed RNA polymerase II] <=> ADP + [DNA-directed RNA polymerase II] phosphate : catalyzed by CDK9 EC 2.7.11.23.
TFIIF and FCP1 cooperate for RNAPII recycling. FCP1, the CTD phosphatase, interacts with RNA polymerase II. Transcription is regulated by the state of phosphorylation of a heptapeptide repeat.The nonphosphorylated form, RNAPIIA, is recruited to the initiation complex, whereas the elongating polymerase is found with RNAPII0. RNAPII cycles during transcription. CTD phosphatase activity is regulated by two GTFs (TFIIF and TFIIB). The large subunit of TFIIF (RAP74) stimulates the CTD phosphatase activity, whereas TFIIB inhibits TFIIF-mediated stimulation. Dephosphorylation of the CTD alters the migration of the largest subunit of RNAPII (RPB1).

• 5' capping

The carboxy-terminal domain is also the binding site of the cap-synthesizing and cap-binding complex. In eukaryotes, after transcription of the 5' end of an RNA transcript, the cap-synthesizing complex on the CTD will remove the gamma-phosphate from the 5'-phosphate and attach a GMP, forming a 5',5'-triphosphate linkage. The synthesizing complex falls off and the cap then binds to the cap-binding complex (CBC), which is bound to the CTD.

The 5'cap of eukaryotic RNA transcripts is important for binding of the mRNA transcript to the ribosome during translation, to the CTD of RNAP, and prevents RNA degradation.

• Spliceosome

singular U SARNP U4U6 di O US CA Spliceosome assembly Us SnRNP recycling Spiceosome 2 Q Splicing catalysis

Fig : Spliceosomal splicing cycle

The carboxy-terminal domain is also the binding site for spliceosome factors that are part of RNA splicing. These allow for the splicing and removal of introns (in the form of a lariat structure) during RNA transcription.

• Mutation in the CTD

Major studies in which knockout of particular amino acids was achieved in the CTD have been carried out. The results indicate that RNA polymerase II CTD truncation mutations affect the ability to induce transcription of a subset of genes in vivo, and the lack of response to induction maps to the upstream activating sequences of these genes.

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