1a.Site specific recombination mediated by the integrase enzyme encoded by integ
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Question
1a.Site specific recombination mediated by the integrase enzyme encoded by integrons and RecA-mediated homologous recombination share a common intermediate structure known as a Holliday junction. However they differ significantly in terms of proteins involved and the outcomes of recombination. Discuss the differences and similarities between these two forms of recombination.
1b.In eukaryotes, chromosomal DNA is complexed with histone proteins to form chromatin. Discuss the structure of the histone-DNA complexes, the various modifications that can occur to the histone proteins and the effects of histones on DNA transcription.
2a.How does the process of transcription differ between prokaryotes and eukaryotes?
2b.You want to investigate the effect of a probiotic on your gut microbiome- the population of bacteria living in your digestive tract. You collect faecal samples prior to and after consumption of the probiotic. Describe in detail how you would sequence the metagenome of the bacteria in these faecal samples and what bioinformatic analyses you would undertake.
Explanation / Answer
1a. The DNA complexed with the histone proteins are called as nucleosomes. A nucleosome contains about 8 histone proteins and 146 bp of DNA (and very rarely 147 bp). These histones are alkaline proteins that help in attaining compact structure of the DNA thereby helping a large chromatin to fit inside the nucleus.
Strucure of Nucleosomes:
146bp of double helix DNA is wrapped around the histone octamer.
The histone octamer is formed by four histone heterodimers (H2A-H2B and H3-H4). The first step in nucleosome assembly is the formation of tetramer, where the H3 and H4 heterodimers pairs to form a tetramer. Further, the H2A and H2B heterodimers binds to the H3-H4 tetramer. During this octamer formation, the H2B and H4 histone folds are joined.
Two popular models have been hypothesised in the chromatin formation: the solenoid and the zigzag formation.
The linker DNA is a part of the chromatin between two nucleosomes (DNA and the histones), also known as non-nucleosome DNA.These linker DNA spans between 20-90 bp. In the solenoid model, the DNA linker is known to be bent and connects two adjecent cores of the nucleosome. While in the zigzag model, there is a straight DNA linker which connect opposite nucleosome thereby forming a zigzag structure.
The different modifications found in histones are
a. Lysine methylation
b. Lysine acetylation
c. Arginine methylation
d. Arginine citrullination
2.b The name prokaryotes means "primitive nucleus". While, the eukaryotes on the other hand are known to have "true nucleus". This suggest that, the primitive nucleus or the prokaryotes have no membrane bound organels and the eukaryotes have the membrane bound organels.
Since the nucleus in prokaryotes are non-membrane bound, the transcription and translation process takes place simultaneously. In the case of eukaryotes, where the nucleus is membrane bound, the transcription process happens inside the nucleus and the translation process happens outside the nucleas (in the cytoplasm). Hence,the transciption process in eukaryotes is complex in comparison to the prokaryotes.
In the prokaryotes, single DNA dependent RNA polymerase are involved in transcription. The transcription process is widely divided into initiation, elongation and termination.
Intiation: An holoenzyme (Sigma factor along with RNA polymrase core enzyme) recongnises specific regions on the DNA to intiate a transcription process. These regions are called as promoter site. In prokaryotes, there are two (TATA box/Pribnow box and -35 sequence) promoter regions.
Elogation: Once the promoter region is recognized, the sigma factor of the holoenzyme leaves the RNA polymerase and thus transcription begins.
Termination: The process of termination in prokaryotes is divided into, Rho dependent and Rho independent. In the case of Rho dependent, the rho factor binds to the newly synthesised RNA (most often) and further terminates the transcription process. While in the case of Rho-independent process, the newly synthesised RNA forms a hair pin loop structure (with the help of palindromes) and further causes termination.
Transcription in eukaryotes:
Unlike prokaryotes, the RNA polymerase in eukaryotes are classified into RNA polymerase I, RNA polymerase II and RNA polymerase III.
Initiation: The promoter site in eukaryotes are known as Hogness box or CAAT box. In eukaryotes in addition to the RNA polymerase, Transcription Factors (TFs) are involved in binding to the promoter region.
Elongation: Similar to prokaryotes, the elongation process occurs from 5' to 3' sequence. During elongation process, the transcription machinery helps in removing the histone proteins present in the DNA thus helping to form a bubble of unwound DNA.
Termination. The RNA polymerase I and RNA polymerase III help in termination process.
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