Question

The majority of eukaryotic genes lack recognizable transcription initiation elements. How do you imagine transcription begins at the correct transcriptional start site when these elements are absent?

Answer 1

Eukaryotic transcription is regulated by a large number of proteins, ranging from sequence-specific DNA binding factors to chromatin regulators to the general transcription machinery and their regulators . Their collective function is to express a subset of genes as dictated by a complex interplay of environmental signals that is only partly understood. Classical biochemistry and cleverly devised genetic screens have led to discoveries of important components of the transcription machinery, and have provided insight into mechanisms involved in transcription by RNA Polymerase II (Pol II). Recent genome-wide expression profiling and factor location profiling have imbued our understanding of the organization of the transcription machinery and nucleosomes throughout the genome. The prevailing view of transcriptional activation is that many sequence-specific regulators interact with their cognate DNA motifs in response to cellular signals. They recruit transcriptional coactivators to alter the local chromatin environment and facilitate assembly of the pre-initiation complex (PIC), which is composed of the general transcription factors (GTFs) and Pol II.

ChIP-chip and ChIP-seq identify the location and level of a protein binding anywhere in any genome :-

Chromatin immunoprecipitation (ChIP) has become an invaluable tool for mapping protein interactions along genomic DNA in vivo and thus has been the single most informative assay in assessing the assembly of proteins on DNA in vivo. A key feature of the ChIP assay is that it preserves physiologically relevant interactions in the cell through formaldehyde crosslinking. Formaldehyde is an ideal crosslinking agent because: i) it quickly permeates the cell and traps native interactions before the cell mounts a physiological response, ii) its single-carbon crosslinker length efficiently generates protein-DNA crosslinks in vivo (protein-protein and protein-RNA crosslinks are also formed), and iii) its readily reversible crosslink is important for subsequent DNA detection methods.

Since its inception , ChIP coupled to microarray detection (ChIP-chip) has proven to be a powerful tool in understanding the interplay of the transcription machinery and chromatin . It can determine the occupancy level of essentially any crosslinkable and immuno-purifiable protein across an entire genome. Early ChIP-chip microarrays have had two important limitations. First, the fabrication of such microarrays has required a sequenced genome. Second, spatial resolution of binding along a genome was limited by probe length and spacing. Today, this has been largely alleviated in the highly tiled (probes every 5–40 bp) second-generation microarrays . Recent break-throughs in cost-effective whole-genome shotgun sequencing has also eliminated the first limitation.

Detection of genomic segments bound by a protein has recently been taken to another level of resolution by coupling the ChIP assay with massively parallel DNA sequencing, called ChIP-seq . The mapping of nucleosome positions across genomes was one of the first applications of ChIP-seq. In the past few years, ChIP-seq has produced whole-genome nucleosome maps for yeast , fly , worm , and human . Others have used ChIP-seq to map the locations of transcription factors .

• Eukaryotes require transcription factors to first bind to the promoter region and then help recruit the appropriate polymerase. RNA Polymerase II is the polymerase responsible for transcribing mRNA.