Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432	Biochemistry	Spring 2002
Lecture Notes:: 3 April	© R. Paselk 2002
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Transcription III

Eukaryotic Promoters and Enhancers

Last time we looked at the promoters for Polymerase I. Let's now look at the promoters for RNA Polymerase II, which is certainly the more interesting enzyme for most.

RNA pol II promoters are more diverse, as would be expected given the vast number of genes it transcribes.

• Constitutive (house keeping) genes expressed in all tissues have on or more copies of the GC box (GGGCGG) or its complement, upstream from the start. It seems to be analogous to the eubacterial promoter.
• Genes specific to tissues often lack the GC box, but have instead an AT rich conserved region 25-30 bp upstream, the TATA box (or Goldberg-Hogness box). The TATA box resembles the Pribnow box of eubacteria, but is at -27 instead of -10 and is not required for transcription. Instead it seems to select the start site.
• Additional promoter sequences for structural genes occur between -50 and -110. These promoters appear to be DNA-binding sites for RNA polymerase and proteins involved in initiation.
  • The CCAAT box is located between -70 and -80.
  • For globin genes the CACCC box is upstream from CCAAT.

RNA Polymerase II also has enhancers - sequences of variable portions and orientation relative to sequences - must be associated with promoters to function.

• need to get full activity of promoter
• seem to be entry points or transcription factor binding sites, either of which enhances RNA polymerase binding.
• So for only associated with tissue specific genes
• Seem to mediate selective gene expression in eukaryotes

RNA Polymerase III: Promoters can be totally within transcribed sequences.

• Binding site for transcription factor that stimulate upstream binding binding of polymerase III promotion can be upstream of start.

E. coli Gene Regulation

Two major types of control: positive and negative.

• Positive control refers to turning on transcription and involves inducers (aka: activators). Two situations occur:
  1. The inducer normally binds to the promoter region of the DNA, stimulating transcription. The presence of a ligand (corepressor) such as a metabolite causes the inducer to be released from the DNA, and transcription is repressed.
  2. The inducer only binds to the promoter region if a ligand (co-inducer) is present, thus promoting transcription.
• Negative control refers to turning off transcription and involves repressors. Two situations occur:
  1. The repressor normally binds to the promoter region of the DNA, blocking transcription. The presence of a ligand (co-inducer) such as a metabolite causes the repressor to be released from the DNA, and allowing transcription to proceed.
  2. The repressor only binds to the promoter region if a ligand (corepressor) is present, thus blocking transcription.

Looked at the binding of the lac repressor to DNA.

Looked at looked at araBAD regulation [overhead]

• Regulatory gene, araC is produced by araBAD operon.
• At low [arabinose] and adequate fuel (low [cAMP]), araC protein dimerizes and binds two regulatory regions of the araBAD operon, araI₁ and araO₂, forming a loop in the DNA encompassing the araO₁, CAP site and araC, regions of the DNA.
• At high [cAMP] in the presence of arabinose
  • arabinose binds to araC protein with the result that the araC protein dimer dissociates and the arabinose-araC protein monomers bind to araI₁ and the adjacent araI₂,
  • cAMP binds to CAP to give a cAMP-CAP dimer which binds to the araC-arabinose proteins, and
  • the resulting complex enhances RNA polymerase binding and RNA synthesis.

Post-transcriptional Processing of RNA

The eubacteria, such as E. coli use mRNA molecule transcripts with no modification, in fact translating them into protein before they are even released from the DNA. (at least some archibacteria have introns, so must have post-transcriptional processing).

Eukaryotes, on the other hand, employ extensive post-transcription processing of their mRNA.

Eukaryotic transcripts are also monocistronic. That is, they have a single gene product coded within each transcript, unlike the multicistronic operons in eubacteria. However, the eukaryotic transcripts are generally much longer than required to code for their single gene product!

The eukaryotic transcript has a number of characteristics:

• They are "capped" with 7-methylguanocine via a 5'-5' triphosphate bridge which is added before the transcript is 20 nucleotides long. (Note that the 7-methylguanosine is essentially put on "backwards" in this cap.)
  • The first two bases of the transcript may also be O^2' methylated (i.e. have methylribose).
  • If the first base is adenosine it may be N⁶ methylated.
  • The capping prevents 5' exonucleases from degrading the mRNA.
  • The cap defines the start of translation.
• A well defined 3' end is determined by enzymatic cleavage and a poly(A) tail is added. Recall that the eukaryotic mRNA transcript is terminated at an approximate location, not a precise base as is the case with eubacteria. The 3' end is created in two steps by the Poly(A) polymerase complex:
  • The end of the transcript is determined by a post-transcriptional cleavage at a point 15-25 nucleotides past a conserved AAUAAA sequence but less than 50 nucleotides before a less conserved U- or GC-rich sequence.
  • The polymerase then adds up to about 250 As via stepwise addition from ATP. The poly(A) polymerase adds the As immediately after the cleavage and without the complex dissociating from the RNA, thus preventing 3' nucleases from attacking the messenger.
    • The poly(A) tail appears to protect the 3' end from degradation, accounting for the long half-lives (hours to days) typical of eukaryotic mRNAs. Not all mRNAs are polyadenylated however. Thus histone mRNAs are not and have short half-lives (<30 min).

Exons and Introns

Eukaryotic transcripts are very heterogeneous, ranging from less than 2000 to >20,000 bases in length. Very little of this hnRNA (heteronuclear RNA) leaves the nucleus and goes to the cytosol for translation however, most is degraded in the nucleus, though the capped end and poly(A) tail both appear in the cytosol!

Thus internal sequences in the hnRNA transcript must be removed before the mRNA leaves the nucleus. The saved segments are referred to as exons while the discarded segments are called introns.

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Last modified 8 April 2002