Eukaryotic Transcription

  The process of eukaryotic transcription is separated into three phases, initiation, elongation, and termination. It is a complex process involving various cell signaling techniques as well as the action of many enzymes. The following information is a detailed description of eukaryotic transcription.


  The transcription of RNA requires the use of three polymerase enzymes, RNA polymerase I, RNA polymerase II, and RNA polymerase III.  Polymerase I is responsible for the transcription of ribosomal RNA; polymerase II is responsible for the transcription of mRNA; and polymerase III is responsible for the transcription of both ribosomal RNA and transfer RNA (8).  While the function of each RNA polymerase enzyme differs, their structures are homologous.  RNA polymerase II is composed of ten subunits, all ten of which are identical or homologous to the subunits in polymerase I and polymerase III (3).  RNA polymerase is comprised of twelve subunits summing a mass of greater that 0.5 MD (2).  Major domains directly involved in transcription are RbpI and Rbp2.  Rbp1 is the largest subunit and contains the active site and Rbp2 contains the hybrid-binding region.  Together these structures combine in a single fold that forms the active center of the enzyme (2).  The surface charge of polymerase II is almost completely negative, with the exception of the positively charged lining of the active center (2).    The active site of the enzyme contains two magnesium ions which stabilize the transition state during the phosphodiester bond formation throughout RNA synthesis (2).  This structure of RNA polymerase II is fundamental in understanding the transcription process.While transcription of ribosomal RNA and transfer RNA are vital processes within organisms, the following transcriptional information is based on the synthesis of messenger RNA using RNA polymerase II.


  The initiation process requires the association of RNA polymerase II with seven general transcription factors, which include TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIJ (8).  Research has shown that TFIID is the only one to have a specific promoter binding ability and in higher eukaryotes, it has the proper polypeptide to bind to the TATA binding protein (8).  Initiation begins with TFIID recognizing the TATA element on the DNA strand.  TFIID binds to the TATA sequence, and this is followed by the assembly of the other transcription factors and RNA polymerase II.  The complex forms the preinitiation complex (PIC) (6).  The interactions the complex makes with the DNA strand makes it difficult for the polymerase to move along the strand.  Therefore, in order for transcription to proceed, the PIC must be dismantled, which requires hydrolysis of ATP by XPB DNA helicase at the subunit TFIIH (6).  This hydrolysis changes the conformation of the PIC, enabling the RNA polymerase to open base pairs around the transcription start site on the DNA strand (7).  The open complex then enters the active site of RNA polymerase II, which is where the base pairs are complemented to the template DNA strand during elongation (7). 


  Once the DNA strand is properly positioned in the metal ion active site of polymerase II, elongation begins.  The elongation phase of transcription is broken down into three stages: promoter escape, promoter-proximal pausing, and productive elongation (7). 

  The first stage of elongation, promoter escape, involves maturing the RNA polymerase II molecule so it is capable of staying in contact with the DNA template strand throughout productive elongation.   During this stage, the polymerase breaks its contact with some of the promoter sequence elements and promoter-bound factors while tightening its hold on the elongating RNA molecule (7).  Once the growing RNA molecule forms a stable association with the transcription complex, the next stage of elongation, promoter-proximal pausing, begins (7).  This relatively stable complex that enters the next promoter-proximal pausing is known as the early elongation complex (EEC) (7). 

  During the promoter-proximal pausing stage, the polymerase II enzyme pauses in the 5’ region of the transcription complex, and upon the appropriate signals, it progresses to productive elongation.  Research has shown that this stage plays a significant role in transcription regulation, and it also serves as a checkpoint before productive elongation (7).  The release from the promoter-proximal pausing stage into the productive elongation stage can occur very quickly; however, the polymerase can remain in the paused stage for a substantial amount of time as well.  Examples of genes that harbor paused polymerases are the mammalian proto-oncogenes FOS and MYC (7).  Generally, this stage is highly involved in transcription regulation and polymerase preparation for elongation.

  Productive elongation is the final stage in the elongation phase of transcription.  During this stage, polymerase II moves through the rest of the gene being transcribed.  The metal ion active site provides a binding for the phosphate group between nucleotide at the 3’ end of the RNA and the adjacent nucleotide, which are designated -1 and +1 respectively (3).  Position +1 in the transcription complex is the position in which ribonucleotides are added to the growing RNA molecule.  Upon the addition of a ribonucleotide, the polymerase II enzyme undergoes translocation to put the next template base in the proper position, with an empty nucleotide binding site at the end of the RNA molecule (3). 

  The specificity for adding a ribonucleotide rather than a deocxyribonucleotide can be attributed to the recognition of the ribose sugar as well as the hybrid RNA-DNA double helix.  Throughout elongation, the transcribed RNA molecule is proofread to ensure no deoxynucleotides or incorrect bases are inserted (3).  After the RNA has elongated through the length of the gene, it reaches termination signals and the transcribed RNA is released.


  Termination follows elongation and is a crucial step in the transcription process.  Many consequences are possible if polymerase II never responds to termination signals.  For example, failure to end elongation can lead to a reduction in the expression of a down stream gene by interfering with the initiation complex of that gene.  It could also obstruct the correct transmission of eukaryotic chromosomes.  Research on termination mechanisms points to two sequences that are required for proper termination.  The first is the poly(A) sequence, AATAAA, and the second is a series of stop sites that are located hundreds of bases downstream.  While both of these sequences make only small contributions, they are both essential in the termination process (5).

RNA Polymerase II Structure

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