Question

1. You are working on an experiment in yeast and wish to know which polymerase transcribes your favorite gene. Your species cannot be transformed- so you can’t make mutations in the polymerases or alter the genome in any way.

a. Briefly describe how you could determine which polymerase transcribes your gene.

b. Describe the experimental results that would indicate it is transcribed by a Class I gene (2pts).

2. For each of the following: define it, for proteins provide their function. Remember, providing an example is not a definition (1 pt each).

PTEF-b

Gdown1

Nucleosome free region

Bromodomain

H2A.Z

H3.3

Answer 1

Ans.1.(a)

• Genetic information encoded in DNA of yeast cells is transcribed into RNA by RNA polymerase II. RNA Pol II transcribes all the protein coding gene as it is highly modulated at individual gene and regulates the homeostasis of cell and programmed development of organism.
• This process is regulated by the combinatorial molecular interactions of transcription factors and with specific DNA sequences at each gene.
• Most of these factors are proteins are the major factors which helps to enable Pol II to gain access to the gene's promoter and to the initiate RNA synthesis at the transcription start site of the gene, and helps in generating a elongate transcription complex that produces a full-length RNA transcript.

Ans 1(b)

DNA sequences in specific gene promoters provides the code which can tell the level of specific genes to be transcribed. Code is mainly of three parts: core promoter, region proximal to the core promoter, and distant enhancer sequences. The core promoter sequence targets the assembly of distinct preinitiation complexes (PICs) which are composed of general transcription factors. Promoter-proximal regions and distant enhancer sequences directs binding of specific transcription factors which are called activators or repressors. Some co-regulators interacts directly with Pol II and GTFs, and influences the expression of gene. Others reorganize the nucleosomes and change the chromatin architecture of the gene this influences the transcription-factor associations and the status of transcription of Pol II.

Ans 2.

• PTEF b-: is a positive transcription elongation factor (P-TEFb), which plays role in the regulation of transcription by RNA polymerase II (Pol II) in eukaryotes. By the experiments it has been identified as a pure factor which needed for the generation of long run off transcripts using in vitro transcription system derived from Drosophila cells.
• G down 1: GDP (G- down protein) when bounds to the alpha subunit, the alpha subunit remains bound to the beta-gamma subunit to form an inactive trimeric protein.
• Nucleosome free region: Nucleosome free regions (NFRs) in promoters are explained by TF (transcription factor) binding. There are only 10–20 TFs make a significant contribution in explaining NFRs, and these TFs are highly enriched for directly interacting with chromatin modifiers.
• Bromodomain: A bromodomain is 110 amino acid protein domain which can recognize acetylated lysine residues, such as N-terminal tails of histones.
• H2A.Z: It is Variant of histone H2A which replaces the conventional H2A in subset of nucleosomes. These histones play a role in transcription regulation, DNA repair, DNA replication and chromosomal stability.
• H3.3: It is a conserved histone variant which is structurally close to the histone associated with active transcription. It plays role in histone replacement at active genes, participate in the epigenetic transmission of active chromatin states.