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Overview of Transcriptional Control
This Sqadia video is the demonstration of Transcription Control of Gene Expression. Activator proteins bind to specific DNA control elements in chromatin and interact with multiprotein coactivator machines, such as mediator, to decondense chromatin and assemble RNA polymerase and general transcription factors on promoters. Alternatively, repressor proteins bind to other control elements to inhibit initiation by RNA polymerase and interact with multiprotein corepressor complexes to condense chromatin. Recombinant DNA techniques are used to prepare a series of DNA fragments that extend from the 5’-untranslated regions of a gene. The DNA fragments are ligated into a reporter plasmid. The DNA is transformed into E. coli to isolate plasmids. The results of this hypothetical example indicate that the test fragment contains two control elements. The 5’ end of one lies between deletions 2 and 3; the 5’ end of the other lies between deletions 4 and 5. The HindIII, XmaIII, and SmaI restriction fragments that encompass the initiation site were individually incubated with a nuclear extract prepared from cultured cells and 32P-labeled ribonucleoside triphosphates. The run-off transcripts synthesized from each fragment were then subjected to gel electrophoresis and autoradiography to determine their exact lengths. In vivo transfection assay measures transcription activity to evaluate proteins believed to be transcription factors. The assay system requires two plasmids. One plasmid contains the gene encoding the putative transcription factor (protein X). The second plasmid contains a reporter gene (e.g., lacZ ). By use of plasmids encoding a mutated or rearranged transcription factor, important domains of the protein can be identified.
RNA polymerases initiates transcription of most genes at a unique DNA position lying upstream of the coding sequence. The base pair where transcription initiates is termed the transcription-initiation start site (TSS). Various proteins (RNA polymerase, activators, repressors) interact with DNA at the promoter or several kilobases distant to the promoter to regulate transcription initiation. The DNA sequences (response elements) that bind regulatory proteins are Cis acting sequences. The regulatory proteins are generally coded by a different gene; hence they are Trans-acting factors. Promoter proximal elements and enhancers (often distal) often are cell-type specific, functioning only in specific differentiated cell-types. Each gene can be regulated by many different control elements. RNA Pol II binding site is the site where the RNA polymerase pre-initiation complex (PIC) assembles.
Control of Transcription - I
Activators can acetylate and demethylate chromatin to yield opened/loosened euchromatin. Opened chromatin can be acetylated by other activators. Repressors can deacetylate chromatin. Decondensed chromatin is open for binding transcriptional activators and Pol II complex. Activators can mediate increased Pol II pre-initiation assembly. Activators can then mediate increased transcription rate of the Pol II (through CTD phosphorylation). Repressors block the above processes. The cofactor proteins that are recruited by DNA bound gene regulators are called co-repressors and co-activators. They have enzymatic activity and can modify other proteins. The modifications typically are methylations, acetylations, or phosphorylations. Ordered binding and interaction of activators and coactivators lead to transcription. Remodelling complexes use ATP to open the chromatin and expose histone tails. Mediator complexes enhance preinitiation complex assembly. Multiple copies of RAP1 bind to a simple repeated sequence at each telomere region, which lacks nucleosomes. This nucleates the assembly of a multiprotein complex through protein-protein interactions between RAP1, SIR2, SIR3, SIR4, and the hypoacetylated N-terminal tails of histones H3 and H4 of nearby nucleosomes. Association of several condensed telomeres forms higher-order heterochromatin complexes. Histones are lightly cross-linked to DNA in vivo using a cell-permeable, reversible, chemical crosslinking agent. DNA in the immunoprecipitated chromatin fragments is released by reversing the cross-link and then is quantitated using a sensitive PCR method.
Control of Transcription - II
In mechanism of histone deacetylation and hyperacetylation in yeast transcription control there is Repressor-directed deacetylation of histone N-terminal tails. The DNA-binding domain (DBD) of the repressor UME6 interacts with a specific upstream control element (URS1) of the genes it regulates. In activator directed hyperacetylation of histone N-terminal tails, the DNA-binding domain of the activator GCN4 interacts with specific upstream activating sequences (UAS) of the genes it regulates. Repression and activation of many genes in higher eukaryotes occurs by similar mechanisms. In ordered binding and interaction of activators and co-activators leading to transcription of the yeast HO gene, the HO gene is initially packaged into condensed chromatin. Activation begins when the SWI5 activator binds to enhancer sites 1200–1400 base pairs upstream of the start site and interacts with the SWI/SNF chromatin-remodelling complex. Binding sites for the five activators required for transcription of TTR in hepatocytes are indicated. The complete set of activators is expressed at the required concentrations to stimulate transcription only in hepatocytes. The yeast two-hybrid system provides a way of screening a cDNA library for clones encoding proteins that interact with a specific protein of interest. This is a common technique for screening a cDNA library for clones encoding proteins that interact with a specific protein of interest.
Transcription Initiation Complex
In the absence of hormone, the receptor is kept in the cytoplasm by interaction between its ligand-binding domain (LBD) and inhibitor proteins. When hormone is present, it diffuses through the plasma membrane and binds to the ligand-binding domain, causing a conformational change that releases the receptor from the inhibitor proteins. The TAR element in the HIV transcript contains sequences recognized by Tat and the cellular protein cyclin T. Cyclin T activates and helps position the protein kinase CDK9 near its substrate, the CTD of RNA polymerase II. UAF and CF, both multimeric general transcription factors, bind to the upstream element (UE) and core element, respectively, in the promoter DNA. Both tRNA and 5S-rRNA genes contain internal promoter elements located downstream from the start site and named A-, B-, and C-boxes. Assembly of transcription initiation complexes on these genes begins with the binding of Pol III-specific general transcription factors TFIIIA, TFIIIB, and TFIIIC to these control elements.