ANDREWS UNIVERSITY
College of Arts and Sciences
Department of Chemistry and Biochemistry
BCHM 422 Biochemistry II
Chapter Twenty Five Lecture Guide
NAME ____________________________
Thought for the Chapter
“I tell you the truth,” he said, “this poor widow has put in more than all the others. All these
people gave their gifts out of their wealth; but she out of her poverty put in all she had to
live on.” Luke 21: 3,4
Outline
Types of RNA
RNA polymerase
Template binding
Chain elongation
Chain termination
Eukaryotic transcription
Polymerases
Promoters
Factors
Posttranscriptional Processing
Messenger RNA
Ribosomal RNA
Lecture
Types of RNA
All cellular RNA is transcribed from DNA templates by RNA polymerases, which create
mRNA— messenger RNA; direct protein synthesis
rRNA— ribosomal RNA; used to interact with DNA or RNA in partnership with proteins
tRNA— transfer RNA; carries amino acids to ribosomes to elongate growing proteins
RNA polymerase
Template binding
Specific locations for RNA transcription must be located on the DNA (template binding)
A single strand of RNA is created in exact image of the “sense” strand. (T becomes U)
Thus the RNA copy is matched against the “antisense” strand of the specific DNA
It is conceivable that the sense strand codes for a different gene matching the “antisense”
strand.
The template, for prokaryotes, contains
Regulatory and control genes: used to control transcription
BCHM 422 Biochemistry II, Chapter Twenty Five Lecture
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Structural genes: codes for genes that ultimately produce proteins
operon: a group of genes transcribed in sequence
polycistronic mRNA: the mRNA arising from a multigene operon (prokaryotic)
“cistronic”: synonym for gene
monocistronic: single gene(eukaryotic)
Promoters (prokaryotic)
The template is located by the RNA polymerase finding promoter sites (initiator sites) on
the prokaryotic chromosome.
Two sites “upstream” of initiation site (+1)
-10 region (Pribnow box/TATA box)
-35 region
The actual sequence will vary. Mutations can increase or decrease transcription.
“Tightness” of binding (K~10-14 M) is responsible for transcription rate variation. If you
have tight binding, it is duplicated a lot.
Holoenzyme (core enzyme (RNA pol) with sigma unit) binds DNA loosely and
nonspecifically. Sigma unit is specific for particular promotor region.
Once holoenzyme binds, sigma unit is released and core binds tightly.
Cell has a complement of sigma factors
Sigma factors determine which genes get transcribed
Chain elongation
RNA polymerase is
Processive – it goes continuously (uninterrupted)
Rapid
Two Kinds of enzymes necessary:
constitutive enzymes (constant low level for cell housekeeping)
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inducible enzymes (synthesized at variable rates depending on circumstances)
negatively and positively supercoiling the DNA (controlled by Topoisomerase s/gyrases)
Chain termination
Two common features
Series of 4-10 consecutive A-T, terminates just past this.
G+C –rich series region just before A-T region
These create structural features in RNA with a “hair-pin” turn. This destabilizes the RNA
polymerase and helps it to fall off.
This slows the RNA polymerase.
Actual termination efficiency depends on:
Above structures
Concentration of dNTPs
Supercoiling of DNA
Salt concentration (in vitro artifact? – possible doesn’t happen in vivo)
Not all sites have the above characteristics
Some sites require a who factor to terminate
1. Enhances termination sites of spontaneous sites (Inherent in structure of DNA that
says stop)
2. Required for non-spontaneous sites
3. May have something to do with DNA/RNA winding/unwinding
(inhibit/accelerate)
Transcription in Eukaryotes
Multiple RNA polymerases
Much more complicated control sequences
BCHM 422 Biochemistry II, Chapter Twenty Five Lecture
RNA polymerases
RNA Polymerase I- in nuclei (nuclear body within nucleus) for most rRNA
RNA Polymerase II- nucleoplasm for most mRNA
RNA Polymerase III- nucleoplasm 5S rRNA, tRNA, cytosolic RNA
These RNA Pols have “compositions of Byzantine complexity”
Promoters (eukaryotic)
RNA Pol I—
Core promoter region (-31 to +6 region)
with required Upstream promotor element (-187 to –107)
RNA Pol II— (structural genes – genes responsible for protein synthesis)
More diversity
Look for GC box upstream
Look for TATA box (25-30 upstream) roughly similar to –10 prokayotic site
Deletion of TATA box not fatal, but initiation site will vary without it
Promotor region
Look for CCAAT box (-70 to –90 upstream); a promotoerregion
Enhancers and silencers exist many hundreds or 1000’s of bp upstream
Activators or repressors can act directly on the enhancers & silencers.
RNA Pol III—
Can exist WITHIN the transcribed region
Transcription factors (Factors that tell where to begin)
Similar to  factors in prokaryotes but much more complicated.
Required for initiation of transcription
Six general factors exist (called general transcription factors - GTFs)
Required for synthesis of all mRNA
Transcription begins with formation of PIC (preinitiation complex), which consists of:
1. TBP (a component of TFIID) binds to SS DNA TATA box
2. TFIIA and TFIIB bind
3. TFIIF binds to RNA Pol II, bringing it to complex formed by 1 & 2
4. TFIIE and TFIIH complete the PIC
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BCHM 422 Biochemistry II, Chapter Twenty Five Lecture
TBP may be only universal TF (found in all species)
Above model is preliminary
Posttranscriptional Processing.
Eukaryotic mRNA is heavily modified following its transcription.
mRNA processing
Poly A tails
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BCHM 422 Biochemistry II, Chapter Twenty Five Lecture
Introns and Exons
Eukaryotic genes have intervening sequences that are not translated into proteins.
Exon-Intron splicing
Two stage process
Requires consensus sequence at the intron-exon junction
1.
2.
3.
Proteins required for this splicing
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BCHM 422 Biochemistry II, Chapter Twenty Five Lecture
Introns are spliced in 5’ to 3’ order
mRNA editing
)
rRNA processing
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BCHM 422 Biochemistry II, Chapter Twenty Five Lecture
Prokaryotes
Uses endonucleases RNase III, RNase P, RNase E and RNase F.
No introns/exons of course
Eukaryote
Only a few rRNAs have introns, but they
catalytically self-splice (RNA world,
Thomas Cech)
1.
2.
3.
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