Transcript Foundations of Biology - Geoscience Research Institute

Ecclesiastes 3:1
1 To every thing there
is a season, and a
time to every purpose
under the heaven:
©2000 Timothy G. Standish
Transcription:
Concentrating on Prokaryotes
Timothy G. Standish, Ph. D.
©2000 Timothy G. Standish
All Genes Can’t be Expressed
At The Same Time
Some gene products are needed by all cells all
the time. These constitutive genes are
expressed by all cells.
Other genes are only needed by certain cells or
at specific times, expression of these inducible
genes is tightly controlled in most cells.
For example, pancreatic b cells make insulin
by expressing the insulin gene. If neurons
expressed insulin, problems would result.
©2000 Timothy G. Standish
Logical Expression Control Points
Increasing cost
DNA packaging
Transcription
RNA processing
mRNA Export
mRNA masking/unmasking and/or
modification
mRNA degradation
Translation
Protein modification
Protein transport
Protein degradation
The logical
place to
control
expression is
before the
gene is
transcribed
©2000 Timothy G. Standish
Introduction
The Central Dogma
of Molecular Biology
Cell
Transcription
Translation
Reverse
transcription DNA
mRNA
Ribosome
Polypeptide
(protein)
Stages of Transcription
1.
2.
3.
4.
l
Transcription can be logically divided
into four distinct stages:
Template recognition
Initiation
Elongation
Termination
Each stage may participate in
regulation, but template recognition and
termination appear to be major players
©2000 Timothy G. Standish
General Model
For
Transcription
Core Enzyme
aa b
s
b
RNA Polymerase
Holoenzyme
©2000 Timothy G. Standish
General Model For Transcription
aa b
s
b
©2000 Timothy G. Standish
General Model For Transcription
aa b
s
b
©2000 Timothy G. Standish
General Model For Transcription
aa b
s
b
©2000 Timothy G. Standish
General Model For Transcription
s
TopIsomerase
I
a
NusAa b
b
Gyrase
©2000 Timothy G. Standish
General Model For Transcription
s
a
NusAa b
TopIsomerase
I
b
Gyrase
Ribosome
Exonuclease
Ribosome
©2000 Timothy G. Standish
General Model For Transcription
s
a
NusAa b
Ribosome
Exonuclease
b
Ribosome
©2000 Timothy G. Standish
General Model For Transcription
s
a
NusAa b
Exo-
b
Ribosome
nuclease
©2000 Timothy G. Standish
General Model For Transcription
s
a
NusAa b
Exo-
b
Ribosome
nuclease
©2000 Timothy G. Standish
General Model For Transcription
s
a
NusAa b
b
Exonuclease
©2000 Timothy G. Standish
General Model For Transcription
s
a
NusAa b
b
Exonuclease
©2000 Timothy G. Standish
General Model For Transcription
NusA
s
aa b
b
Exonuclease
©2000 Timothy G. Standish
General Model For Transcription
s
a
NusAa b
b
©2000 Timothy G. Standish
RNA Polymerase
1.
2.
3.
4.
5.
RNA Polymerase is a spectacular
enzyme; it performs the following
functions:
Recognition of the promoter region
Melting of DNA (Helicase +
Topisomerase)
RNA Priming (Primase)
RNA Polymerization
Recognition of terminator sequence
©2000 Timothy G. Standish
Prokaryotic Transcription Initiation
The s subunit of prokaryotic RNA
polymerase is necessary for promoter
recognition and binding of RNA
polymerase to the promotor
Different s subunits allow recognition of
different types of promoters; thus the type
of genes transcribed can be modulated by
altering the types of s subunits which attach
to RNA polymerase
©2000 Timothy G. Standish
Prokaryotic Transcription Initiation
RNA
Pol.
s
P1
Heat Shock Gene
P2
Constitutive Gene
Different promoters
©2000 Timothy G. Standish
Prokaryotic Transcription Initiation
RNA
Pol.
s
P1
Heat Shock Gene
P2
Constitutive Gene
Different promoters
©2000 Timothy G. Standish
Transcription Termination
There are two types of termination:
Rho dependent requires a protein called
Rho, that binds to and slides along the RNA
transcript. The terminator sequence slows
down the elongation complex, Rho catches
up and knocks it off the DNA
Rho independent termination depends on
both slowing down the elongation complex
with a hairpin and a U-rich region that
destabilizes the elongation complex
©2000 Timothy G. Standish
Termination
Rho Independent
RNA
Pol.
RNA
Terminator
5’
RNA
Pol.
RNA
5’
©2000 Timothy G. Standish
Termination
Rho Independent
RNA
Pol.
RNA
5’
5’
RNA
Pol.
RNA
Terminator
©2000 Timothy G. Standish
Termination
Rho Dependent
Terminator
RNA
Pol.
RNA
5’
r
RNA
Pol.
RNA
5’
r
The terminator
sequence slows
RNA polymerase
©2000 Timothy G. Standish
Termination
Rho Dependent
Terminator
RNA
Pol.
RNA
5’
r
Help, Rho
hit me!
r
RNA
5’
RNA
Pol.
Rho catches up
with RNA
polymerase
©2000 Timothy G. Standish
Termination
Rho Dependent
Terminator
RNA
Pol.
RNA
5’
5’
r
The elongation
complex disintegrates
RNA
Pol.
RNA
©2000 Timothy G. Standish
©2000 Timothy G. Standish
Transcription
Coding
(sense) strand
5’
3’
3’
5’
Template
(antisense) strand
©2000 Timothy G. Standish
Transcription
Coding
(sense) strand
5’
3’
3’
5’
5’
RNA
Pol.
Template
(antisense) strand
RNA
©2000 Timothy G. Standish
Transcription
Coding
(sense) strand
5’
3’
3’
5’
Template
(antisense) strand
RNA
Pol.
5’
©2000 Timothy G. Standish
Products of Transcription
Transcription produces three major RNA products:
1 Ribosomal RNA (rRNA) - Several rRNAs are vital
constituents of ribosomes
2 Transfer RNA (tRNA) - The molecule that physically
couples nucleic acid codons with specific amino acids
3 Messenger RNA (mRNA) - The nucleic acid messenger
that carries encoded information from genes on DNA to
the protein manufacturing ribosomes
©2000 Timothy G. Standish
Transfer RNA (tRNA)
Acts as the adapter molecule between the genetic code
on mRNA and the protein “language”
75-85 bases long
A specific amino acid is covalently linked at the 3’ end
Elsewhere on the molecule is an anticodon
complimentary to the specific amino acid codon on
mRNA that codes for the amino acid carried by the
tRNA
Contain a number of modified bases
©2000 Timothy G. Standish
A “Simple” Gene
Transcription
Start Site
5’
5’ Untranslated Region
3’ Untranslated Region
Protein Coding Region
3’
RNA Transcript
Promoter/
Control Region
Terminator
Sequence
©2000 Timothy G. Standish
Transcription Initiation
Proteins called transcription factors bind
to the promoter region of a gene
If the appropriate transcription factors
are present, RNA polymerase binds to
form an initiation complex
RNA polymerase melts the DNA at the
transcription start site
Polymerization of RNA begins
©2000 Timothy G. Standish
Initiation
T. F.
Promoter
T. F.
RNA
Pol.
RNA
Pol.
RNA
5’
©2000 Timothy G. Standish
Transcription And Translation
In Prokaryotes
5’
3’
3’
5’
RNA
Pol.
Ribosome
mRNA
Ribosome
5’
©2000 Timothy G. Standish
Heat Shock Response
Elements
Sudden changes in the temperature of cells cause stress
in response to which heat shock genes are expressed
At least some heat shock genes are thought to be
chaperones that help proteins fold correctly
Heat shock genes have Heat Shock Elements (HSEs) in
their control regions
Heat Shock Transcription Factors (HSTFs) bind the
HSEs up regulating expression of heat shock gene
products
©2000 Timothy G. Standish
©2000 Timothy G. Standish
Control of Gene Expression
Cytoplasm
Packaging
Degradation
DNA
Transcription
Transportation
Modification
RNA
RNA
Processing
mRNA G
G
AAAAAA
Nucleus
Export
Degradation etc.
AAAAAA
Translation
©2000 Timothy G. Standish
A “Simple” Gene
Transcription
Start Site
5’ Untranslated Region
5’
3’ Untranslated Region
Protein Coding Region
3’
RNA Transcript
Promoter/
Control Region
Terminator
Sequence
©2000 Timothy G. Standish
Initiation
T. F.
Promoter
T. F.
RNA
Pol.
RNA
Pol.
T. F.
RNA
5’
©2000 Timothy G. Standish
Transcription And Translation
In Prokaryotes
5’
3’
3’
5’
RNA
Pol.
Ribosome
mRNA
Ribosome
5’
©2000 Timothy G. Standish