Humboldt State University ® Department of Chemistry

Richard A. Paselk
Chem 432

Biochemistry

Spring 2002
Lecture Notes:: 27 March

© R. Paselk 2002
  
     
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Transcription, cont.

Last time we looked at initiation and elongation in the transcription of RNA in E. coli. The third part of transcription is:

Termination, again a complex and multi-step process. RNA polymerase is a key player in termination, where the b subunits can both increase and decrease the efficiency of termination. There are two types of termination, one dependent on another protein , the rho (r) factor, and the other on specific termination sites in the DNA of E. coli.

In the case of Termination sites there is not a unique base as a stop point. Rather some common structural features occur which result in termination:

  1. A series of 4 - 10 consecutive A-Ts with As on the template strand - RNA termination occurs in or just past this sequence.
  2. A G+C rich region with a palindromic sequence immediately proceeds the A-T sequence. The RNA transcribed from the G-C rich sequence will be able to form a hair-pin structure (due to the self-complementarity of the palindromic sequence).
  3. A series of 6-8 As on the DNA template strand, coding for Us in the RNA, which will bind to the DNA template only weakly.

The result of this termination site is that the RNA transcript will form a hair-pin which in turn slows the RNA polymerase. Since the last RNA synthesized, and involved in the RNA-DNA double-strand, will be the poly-U stretch, the polymerase and RNA will tend to peal off, as the somewhat more stable A-T bonds displace the A-U bonds, halting synthesis.

Rho factor enables non-spontaneously terminating sites to terminate and increases the efficiency of the spontaneous termination sites discussed above. The rho factor is an ATP dependent helicase, which can unwrap the DNA-RNA hybrid helix. This ring-shaped hexameric protein (six 50 kD subunits) binds to the nascent RNA strand at a C-rich recognition site, then migrates 5'Æ3' towards the polymerase. When the polymerases 'pauses' at a G-C rich termination region, r catches up, unwinding the RNA-DNA double helix and releasing the RNA polymerase resulting in termination.

 

Eukaryotic RNA Polymerases

Unlike prokaryotes, eukaryotes have a variety of RNA polymerases: a mitochondrial polymerase (and a chloroplast polymerase in plants), and three nuclear polymerases. We will focus on the three nuclear RNA polymerases:

  1. RNA Polymerase I: This enzyme is localized in the nucleolus and is responsible for synthesizing the rRNA precursor.
  2. RNA Polymerase II: This enzyme is in the nucleoplasm, synthesizing the mRNA precursors.
  3. RNA Polymerase III: This enzyme is also in the nucleoplasm, but specializes in synthesizing tRNA, the 5s rRNA and other small RNA precursors.

There is much variety and complexity in the make-up of the three polymerases. All are large enzymes with up to 14 different subunits. Polymerase II, which is also know as RNA Polymerase B, has gathered the greatest attention as one would expect. A comparison of these enzymes based on Polymerase II from yeast follows. The subunits of polymerase II are named RPB1-10 (for RNA Polymerase B 1-10).

  1. RNB1 (220 kD): This largest subunit has homologous subunits with similar sequences in polymerases I & III as well as the E coli subunit b'. It has an unusual structural feature not found in prokaryotes, a long C-terminal 'tail' (the CTD = C Terminal Domain) with 27 repeats of the sequence PTSPSYS (pro-thr-ser-pro-ser-tyr-ser). Note that this sequence is quite hydrophilic, and has many potential sites for phosphorylation (5/6 have -OH groups).
  2. RNB2 (150 kD): The next largest subunit again has homologous subunits with similar sequences in polymerases I & III and this time E coli subunit b. As in the case of E coli this subunit binds a NTP. Both RNB1 and RNB2 participate in the catalytic site of the polymerase
  3. RNB3 (45 kD): The next largest subunit is homologous with the E coli subunit a. Two copies are present in the polymerase and are necessary for core assembly, as is the case in the bacteria. It is unique to Polymerase II in eukaryotes.
  4. RNB4 (32 kD): This subunit is the last to have a bacterial homolog, in this case sharing significant sequence similarity with the s factor of E coli and thus thought to be involved with promoter recognition. It readily dissociates from the polymerase. Like RNB3, it is unique to Polymerase II in eukaryotes.
  5. RNB5 (27 kD), RNB6 (23 kD), RNB8 (14 kD), & RNB10 (10 kD) are all shared by the three eukaryotic polymerases.
  6. RNB7 (17 kD) Is unique to Polymerase II, and readily dissociates.
  7. RNB9 (13 kD).

 

Promoters and Enhancers

Eukaryotic polymerases differ in the strategies of promotion.

RNA Polymerase I: There is only one type of rRNA gene in a given species of eukaryote, though there may be hundreds or even thousands of copies of that gene. As a result there is only one promoter in each species for polymerase I, though the promoters are quite species specific.

The rRNA promoter for yeast has a sequence from -31 to +6 (core promoter element) with an additional upstream elements at - 187 and -107. A short sequence is probably required for polymerase binding with the rest required for transcription factors (Nested control regions).

The product of RNA polymerase I is a 7500 bp transcript (approx. 45s) which has, in order ( 5'Æ3') the 18s, 5.8s, and 28s rRNAs separated by spacers.

Pathway Diagrams

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Last modified 28 March 2002