G3 course reading - Tantin
MBIOL 6420
8:30-9:30 ASB 210
Dean Tantin, PhD
X7-3035, [email protected]
Required course reading
For many of you, this is a review of undergraduate material,
but study these slides in advance of the first day of my
lectures. There is much that is testable in here, and things in
the next few lectures will rely on knowing this material.
(Simplified) flow of genetic information…
transcription
DNA
pre-mRNA
mRNA
splicing
The central dogma
Francis Crick
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 (with U in place of T).
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 (in bacteria this can
overlap with a negative element termed the “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.
Transcription is a HEAVILY REGULATED process
• Transcription is frequently regulated both temporally (e.g.,
response to signals, stress) and spatially (i.e., particular DNA
segments transcribed).
• Regulation largely but not entirely occurs prior to transcription
complex assembly and active phosphodiester bond formation.
• This means that the recognition needed for regulation must be of
standard unmelted B-form DNA (or of chromatin, which uses the
underlying DNA sequence to organize itself). Either way, DNA:
see attached review from Coller and Kruglyak (“It’s the
Sequence, Stupid!”)
• Following this line of reasoning, cells must use strategies to
recognize sequences in unmelted DNA to direct when and where
the transcription machinery is active.
The DNA helix and how it is read
22Å
34Å
22Å
Yellow:
zinc finger
protein
recognizing
12Å
DNA…
The DNA helix and how it is read
Specific chemical groups
exposed in the major and
minor grooves provide
information about the DNA
sequence.
The sequence is“read”
through specific chemical
interactions (e.g., hydrogen
bonding) with transcription
factors that fit into these
grooves and can only
recognize the chemical groups
in a specific pattern.
“Wiring diagrams” provide a shorthand for regulation