Question

A cell line isolated from a patient shows decreased transcription of the pS2 gene in response to oestrogen.

1) Explain how you would investigate whether all of the key components for the initiation of transcription are recruited to the pS2 promoter in the right order. Include all of the different classes of proteins that you would look for. [15 marks ]

2) Your results demonstrate that although all of the key components are recruited to the promoter of your gene of interest in the right order to form a complete pre-initiation complex, transcription initiation is very slow. This suggests that one or more components might contain mutations that result in stalled initiation of transcription. Discuss the possible identity of two different mutated components, giving reasons why. [8 marks]

Please help in answer 2) according to the marks needed and with relevant details. Cheers :)

Answer 1

2. In eukaryotes, the term promoter describes all the sequences that are important in the initiation of transcription of a gene. For some genes these sequences not only includes the core promoter (the site at which initiation complex is assembled) but also one or more upstream promoter elements (lies upstream of core promoter region). As given in the question, all of the key components are recruited to the promoter of gene of interest in the right order to form a complete pre-initiation complex, but transcription initiation is very slow. This suggests that one or more components might contain mutations that results in stalled initiation of transcription. The possible identity of different mutated components are:

(i) Mutation in the core promoter region- The core promoter consists of two segments: the -30 or TATA box (consensus 5'-TATAXAX-3' where X is A or T) and the initiator(Inr) sequence (consensus 5'-Py₂CAPy₅ -3') located around nucleotide +1. As TATA box and Inr sequence serve as specific binding sites for general transcription factors and the consensus sequence present in the core promoter primarily determine the location of the startpoint. So, any mutation in the sequences of these boxes led to the weak or non-specific association of the transcription factor and hence the transcription rate will be very slow.

(ii) Mutation in the cis-acting sequences- These sequences serve as the binding sites for a wide variety of regulatory factors that control the expression of individual genes. The cis-acting sequences are frequently located upstream of the TATA box. The two regulatory sequences that are found in many eukaryotic genes are CAAT box and GGGCGG(known as GC box). These two regulatory sequences are orientation independent. The CAAT box is recognized by the activators NF-1 and NF-Y whereas GC box is recognized by Sp1 activator. So, any mutation in these sequences does not allow activators to bind to these sequences and hence alter the rate and frequency of transcription.

Few genes also contain downstream promoter element (DPE) located at position +28 to +32. DPE has a variable sequence and binds TFIID. So, any mutation in these DPE alters the binding of the transcription factor and hence affects the rate of transcription.                                                                                           

(iii) Mutation in the subunits of the RNA polymerase II- As eukaryotic RNA Polymerase II consists of 12 subunits .So, mutation in any of the components of the RNA polymerase II that binds with the transcription factors involved in the initiation affects the binding of the RNA polymerase II with the transcription initiation factors and hence affects the rate of transcription.