Question

1 2 3 4 5 6 7 8 9 lane (fig64) TBP TEIID Pol 11 ΤΕΙΙΗ NELF CDK9 The figure above is a series of ChIP-PCR reactions using antibodies against different promoter protein components and primers for the human Hsp70 promoter, a TATA-box containing promoter. List the six lanes, in order, that represent the steps in which each protein loads on to the promoter. You should start with no proteins bound and end with the proteins bound at the promoter at the beginning of elongation.

Answer 1

Chromatin immunoprecipitation, or ChIP, is an antibody-based technology used to selectively enrich specific DNA-binding proteins along with their DNA targets. ChIP is used to investigate a particular protein-DNA interaction, several protein-DNA interactions, or interactions across the whole genome or a subset of genes.

ChIP utilizes antibodies that selectively recognize and bind proteins, including histones, histone modifications, transcription factors, and cofactors, to provide information about chromatin states and gene transcription. A combination of proteomic analysis and molecular biology techniques used in ChIP allow for the ability to understand gene expression and regulation in cells or tissues of interest.

How does ChIP work?

The principle behind ChIP is relatively straightforward and relies on the use of an antibody to isolate, or precipitate, a certain protein, histone, transcription factor, or cofactor and its bound chromatin from a protein mixture that was extracted from cells or tissues. Hence, the name of the technique: Chromatin Immunoprecipitation. In ChIP-PCR or ChIP-seq, immune-enriched DNA fragments are then able to be identified and quantified using widely available PCR or qPCR reagents and Next Generation Sequencing (NGS) technologies.

ChIP-PCR is performed to analyze histone modifications and/or protein binding to a known subset of target loci in the genome. In ChIP-PCR, immune-enriched DNA fragments are identified and quantified using widely available PCR or qPCR reagents and technologies. Rapid and quantitative comparisons of specific regions within the genome across multiple samples can be achieved using ChIP-qPCR. This is cheaper and more time efficient than whole genome sequencing methods.

The TATA box is a component of the eukaryotic core promoter and generally contains the consensus sequence 5'-TATA(A/T)A(A/T)-3'. In humans only 24% of genes have promoter regions containing the TATA box. Genes containing the TATA-box tend to be involved in stress-responses and certain types of metabolism and are more highly regulated when compared to TATA-less genes. The TATA box is usually located 25-35 base pairs upstream of the transcription start site. Genes containing the TATA box usually require additional promoter elements, including an initiator site located just upstream of the transcription start site and a downstream core element (DCE). These additional promoter regions work in conjunction with the TATA box to regulate initiation of transcription in eukaryotes.

Role in transcription initiation:

The TATA-box is the site of preinitiation complex formation, which is the first step in transcription initiation in eukaryotes. Formation of the preinitiation complex begins when the multi-subunit transcription factor II D (TFIID) binds to the TATA box at its TATA-binding protein (TBP) subunit. The conformational changes induced by TBP binding to the TATA box allows for additional transcription factors and RNA polymerase II to bind to the promoter region. TFIID first binds to the TATA box, facilitated by TFIIA binding to the upstream part of the TFIID complex. TFIIB then binds to the TFIID-TFIIA-DNA complex through interactions both upstream and downstream of the TATA box. RNA polymerase II is then recruited to this multi-protein complex with the help of TFIIF. Additional transcription factors then bind, first TFIIE and then TFIIH. This completes the assembly of the preinitiation complex for eukaryotic transcription.

The human negative elongation factor (NELF) is a four-subunit protein complex that inhibits the movement of RNA polymerase II (RNAPII) at an early elongation stage.

CDK9 stimulates release of paused polymerase and activates transcription by increasing the number of transcribing polymerases and thus the amount of mRNA synthesized per time.

Below is the six lanes in order:-

Lage Lone Lage Lage Lane Lore Lage S 2 6 TBP TBP TBP TBP TBP No protein TBP TEIID TFIID TEIID TFIID poli TAID POLIT Poll TFITH PoliT TFITH TRIIH NELE COKE CS Scanned with CamScanner