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METHOD
1. Set up in vitro transcription
reaction with DNA template,
32P-UTP, plus or minus
transcription factor SP1
2. Separate transcripts by gel
electrophoresis
QUESTIONS:
1. Name the DNA for which
transcription requires
SP1.
2. What is the evidence as
seen in the
autoradiogram?
3. What type of
transcription factor is
SP1?
QUESTIONS:
Based on the results shown in the
autoradiogram, we see a band in
lane 1 (from transfection with
plasmid 1) with but not in lane 2
(from transfection with plasmid
2).
1. What does the band in the
autoradiogram represent?
2. What do these results tell us
about the nature of the extra
piece of DNA included in plasmid
1 and absent in plasmid 2?
(What is the “name” for this extra
piece of DNA?)
Lecture 9
Transcription Termination,
mRNA processing, & posttranscriptional control
Reading:
Chapter 12
Molecular Biology syllabus web site
Transcription termination
• Several mechanisms exist to regulate the termination of
transcription in bacteria and eukaryotic cells
• In bacteria, the two principle mechanisms involve RNA
polymerase and one of these also requires the termination
factor Rho
• In eukaryotes, the mechanisms for terminating transcription
differ for each of the three types of RNA polymerase
Rho-independent termination occurs at
characteristic sequences in E. coli DNA
Premature termination by attenuation helps
regulate expression of some bacterial
operons
Mechanism of attenuation of trp-operon
transcription
Rho-dependent termination sites are
present in some -phage and E. coli
genes
• The Rho factor is a hexameric protein around which a 70- to
80-base segment of the growing RNA transcript wraps
• Rho then moves along the RNA in the 3 direction until it
eventually unwinds the RNA-DNA hybrid at the active site of
RNA polymerase
• Whether transcription is terminated or not depends on
whether Rho “catches up” to RNA polymerase
• Rho-dependent sites have no clear consensus sequence and
Rho-dependent termination operates at relatively few
operons
Figure 11-4
Three eukaryotic RNA polymerases
employ different termination
mechanisms
• RNA polymerase I is terminated by a mechanism that
requires a polymerase-specific termination factor, which
binds downstream of the transcription unit
• RNA polymerase II is terminated in a region 0.5-2 kb beyond
the poly(A) addition site, and termination is coupled to the
process that cleaves and polyadenylates the 3 end of a
transcript
• RNA polymerase III is terminated after polymerizing a series
of U residues
Transcription of HIV genome is
regulated by an antitermination
mechanism
Processing of eukaryotic mRNA
The 5-cap is added to nascent RNAs
after initiation by RNA polymerase II
Pre-mRNAs are cleaved at specific 3
sites and rapidly polyadenylated
During the final step in formation of mature,
functional mRNA, introns are removed and
exons are spliced together
Splicing occurs at short, conserved
sequences
Consensus sequences around 5 and 3 splice sites in vertebrate pre-mRNA
Analysis of RNA products formed in an
in vitro splicing reaction
Splicing proceeds via two sequential
transesterfication reactions
Small nuclear RNAs (snRNAs) assist in
the splicing reaction
Self-splicing introns-evolutionary models of trans-acting snRNAs?
Other post-transcriptional
regulatory mechanisms
• Alternative splicing (e.g. ion channels affecting auditory
cells; affecting wiring/neuronal connections in the brain)
• mRNA 3’ ends that target mRNA to cytoplasmic location
(resulting in protein gradients across cell)
• mRNA stability affected by 3’ untranslated sequences
• Regulation of antisense transcripts and siRNA
• RNA editing (common in mt in protozoa/plants and
chloroplasts; rarer in higher eukaryotes)
• Post-transcriptional modification (e.g. tRNA)
• Translational activation of mRNAs (cytoplasmic
polyadenylation of stored mRNAs with short polyA tailsinduced upon fertilization of xenopus oocytes or synaptic
activity in neuronal dendrites)