Question

The transport of Potassium across a eukaryotic membrane can happen by either a passive or an active transport. The active transport is used when Potassium has to move against the concentration gradient that is from a low concentration outside cells to a high concentration inside cells. Ion channels mediate the passive transport and these transport ions from a high concentration to a low concentration.
The Potassium channel is very selective for the transport of Potassium.
a) Explain how the selectivity of the potassium channel is ensured and why the smaller ion lithium will not be transported through the potassium channel.
The gene encoding the Potassium channel in eukaryotes is transcribed into a pre-mRNA.
b) List the three elements in a pre-mRNA in eukaryotes that is not found in a prokaryotic mRNA?
c) RNA polymerase II is responsible for the transcription of the gene encoding the Potassium channel; the carboxyl-terminal domain of the RNA polymerase II has a special function, describe the function?
d) Give the post-translational modification, which is responsible for the regulation of the activity of the carboxyl-terminal domain of RNA polymerase II.

Answer 1

a) Potassium channel is a tetramer of identical subunits, each of which includes two membrane spanning alpha helices. It is cone shaped with larger opening facing the inside of the cell. The four identical subunits form a selective pore that runs through the center of the potassium channel. A K⁺ ion can fit in the pore with solvated and interact with the channel proteins that eventually reduces the thickness of the membrane and allows solvated ion to enter the membrane. A five amino acid stretch known as the selectivity filter functions in the potassium ion channel by which K⁺ ion interact. This region has a relatively extended conformation and is oriented in such a manner that the peptide carbonyl groups are directed into the channel, facilitating interaction with the K⁺ ions. The selectivity filter is completely permeable to potassium ions but block the flow of other small ions like lithium. The narrow diameter of the selectivity filter of the potassium channel enables the filter to reject ions with radius larger than 1.5 Angstrom.

b) The nascent RNA synthesized by RNA polymerase II is called hnRNA or pre-mRNA. It contains both unwanted base sequences (introns) alternated with useful base sequences (exons). The three things that are happen in an eukaryotic pre-mRNA as compared to prokaryotic pre-mRNA are 5' capping, 3' tailing by polyadenylation and RNA splicing.

c) The carboxy-terminal domain (CTD) of RNA polymerase II consists of 52 repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. It is the portion of the RNA polymerase II that is involved in the initiation of the DNA transcription, the capping of the RNA transcript and attachment to the spliceosome for RNA splicing. Firstly, It serves as a flexible binding scaffold for several nuclear factors, which is determined by the phosphorylation of the CTD repeats to initiate the process of DNA transcription. Secondly, the CTD is also the binding site of cap synthesizing and cap binding complex. In eukaryotes, after transcription of the 5' end of an RNA transcript, the cap synthesizing complex on the CTD remove the gamma phosphate and attach a GMP, forming methy GTP linkage. The synthesizing complex then falls off and the cap then bind to the cap binding complex, that remain bound to CTD. Lastly, The CTD is also the binding site for spliceosome factors that play an important role in RNA splicing. This allow splicing with the removal of introns from the primary transcript.

d) The CTD of the RNA polymerase II is regulated by post-translational modifications that are essential for its proper functioning in order to convert the primary transcript into functional mRNA. The post-translational modification includes phosphorylation of the CTD of RNA polymerase II. RNA polymerase II exist in two forms unphosphorylated and phosphorylated, which are IIA and IIO respectively. The transition between this two forms facilitates different functions for transcription. The phosphorylation of the CTD is catalyzed by a general transcription factor (GTF) named Transcription factor II human (TFIIH) which serve two purposes, firstly to unwind the DNA at the transcription start site and secondly to phosphorylate. The form polymerase IIA joins with the preinitiation complex, more tightly than polymerase IIO form, because of higher affinity to the TATA-box binding protein (TBP). The form polymerase IIO helps in the elongation of the RNA chain. The elongation initiates with the phosphorylation of Serine at position 5 (ser5), by the help of TFIIH. The phosphorylated Ser5 incorporates enzymes like guanyl transferase to cap the 5' end of the hnRNA and 3' tailing factors to polyA sites. CDK8 and cyclin C are components of RNA polymerase II that phosphorylates the CTD. CDK8 regulates transcription by targeting the CDK7/cyclin H subunits of TFIIH, providing a link between the mediator and transcription machinery. With the phosphorylation of the second serine (ser2), elongation is activated. The termination of elongation is achieved by dephosphorylation and when the domain is fully dephosphorylated, the RNA polymerase II is recycled and catalyzes the same process with another initiation site.