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Gene Expression
MBIOL 6640
10:45-11:35 ASB 210
Course director:
Dean Tantin, PhD
X7-3035, [email protected]
Lecture 0
For many of you this is a review of undergraduate material,
but study these slides in advance of the first day of class.
There is much that is testable in here, and things in lectures 14 will blow by you if you are not familiar with this material.
(Simplified) flow of genetic information…
transcription
DNA
pre-mRNA
mRNA
splicing
Protein
translation
Gene expression begins with the process of TRANSCRIPTION
Some terminology (applies to prok. and euk. transcription):
• Transcription is the synthesis of an RNA chain from a DNA
template.
• RNA synthesis proceeds in the 5’3’ direction and is performed
by RNA polymerase and ancillary factors.
• RNA synthesis utilizes NTPs, with uridine triphosphate (UTP) in
place of dTTP in base pairing with adenosine. Thus, RNA is
marked by the presence of a 2’ hydroxyl and uridylate.
• Only one strand of the DNA double helix is utilized as the
physical template for RNA synthesis, and is termed the template
strand.
• The coding strand has a 5’-3’ sequence that is identical to the 5’3’ sequence of the RNA.
Gene expression begins with the process of TRANSCRIPTION
More terminology:
• The promoter is a region of DNA at the 5’ end of the gene that
controls its expression. In eukaryotes it includes the initiation site
(start site), the first base that is transcribed (bacteria=operator).
• The terminator is a DNA sequence that directs where RNA
synthesis stops (this is different from a stop codon). The
intervening DNA is considered the “body” of the gene. A
transcription unit is all RNA synthesized in a single RNA chain.
An “operon” is a bacterial transcription unit that consists of
multiple open reading frames coding for multiple proteins.
• The standard nomenclature for describing the position of DNA
base pairs relative to the start site considers +1 as the start site
and -1 as the base before it (there is no zero). Downstream DNA
is any DNA that is found in the direction the RNA polymerase
travels. The opposite is true for upstream DNA.
Gene expression begins with the process of TRANSCRIPTION
Overview of the prokaryotic transcription cycle
The basic form of the cycle is conserved
in eukaryotes.
Promoter (template) recognition:
Core RNA polymerase cannot find a promoter by itself. It
requires promoter specificity factors, called sigma (s)
factors.
Open complex formation and initiation:
--The DNA is unwound to form the bubble.
--Formation of the first phopshodiester bonds.
2-9 base “abortive transcripts” synthesized.
Promoter clearance (promoter escape):
The transition to processive transcription; the bubble
travels with the polymerase.
Elongation:
Incorporation of ribonucleotides into the RNA chain.
Termination:
Synthesis ends and the RNA is released.
Promoter recognition—bacterial RNA polymerase
Two forms:
• ‘Core’ with the subunit composition
aabb’w (=a2bb’w).
• ‘Holoenzyme’* also includes s.
• There are multiple s factors, so the
polymerase can recognize promoters
with different recognition sequences.
• The most common s factor is s70.
Eukaryotes lack true sigma factors, and
have different strategies for recognizing
promoters.
*‘Holoenzyme’ refers to an active form in
which all the subunits and cofactors
needed for activity are present (as
contrasted with ‘apoenzyme’).
Promoter recognition—bacterial RNA polymerase
b’—largest subunit. Forms part of active site. Interacts nonspecifically
with DNA and nascent RNA.
b—forms the rest of the active site. Interacts nonspecifically with DNA
and nascent RNA.
a—determinants for assembly of RNAP. Recognizes DNA
nonspecifically, OR can recognize a specific Upstream Promoter
element (“UP element”) at certain highly expressed bacterial genes.
w—stabilizes the assembled RNAP.
Sigma factors greatly increase the affinity of polymerase for promoter
DNA (by decreasing the off rate). Holoenzyme bound to DNA that is
unmelted is called the closed complex.