We learned that E. coli terminates transcription using either the -dependent or

Question

We learned that E. coli terminates transcription using either the -dependent or -independent mechanism.

a. How are these two mechanisms different?

b. How are these two mechanisms similar?

c. Suppose you are studying -dependent transcriptional termination in the lab. Propose two ways that you could interfere with the -dependent transcriptional termination process. Think in terms of specific changes you could make to the DNA or mRNA that would inhibit transcriptional termination.

d. Suppose you are studying -independent transcriptional termination in the lab. Propose two ways that you could interfere with the -independent transcriptional termination process. Think in terms of specific changes you could make to the DNA or mRNA that would inhibit transcriptional termination.

Explanation / Answer

a) In -dependent termination, an additional RNA-binding protein called Rho () is required. It binds as a hexameric complex on the RNA and slides along the RNA strand. Once it reaches the RNA polymerase, the protein complex knocks-off the RNA polymerase from the DNA, and terminates transcription.

In -independent termination, also known as intrinsic termination, transcription is terminated without the help of any additional factors. A hairpin loop on the transcribed RNA strand, along with long stretches of T or A on the template strand of DNA, causes the RNA polymerase to pause at the hairpin loop and get stalled, ultimately resulting in its spontaneous dissociation.

b) Both -dependent and -independent mechanisms of transcription termination in prokaryotes, rely on hairpin structures on the transcribed RNA. Apart from their role in -independent transcription termniation, as discussed in part (a), RNA hairpin loops are also required in -dependent termination, allowing the protein to catch up with the RNA polymerase, which being faster than the protein would move ahead, unless the hairpin loop is present to stall it.

c) Two methods to interfere with -dependent termination, by inducing changes in DNA or RNA sequences.

1. Frameshift mutation within the gene encoding protein, thereby blocking the expression and formation of the protein itself.

2. Mutation within the proetin binding sequence on the transcribed RNA, such that the protein cannot bind to the RNA strand

d) Two methods to interfere with -independent termination, by inducing changes in DNA or RNA sequences.

1. Mutation within the template DNA sequence, such that the hairpin loop necessary for the RNA polymerase to stall, is not formed.

2. Changing the nucleotide sequence of the template DNA strand, such that the poly-T or poly-A stretch is not present.

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