| Chem 432 |
Biochemistry |
Spring 2002 |
| Lecture Notes:: 25 March |
© R. Paselk 2002 |
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Transcription
Recall the RNA is:
- ribose based,
- requires A, C, G, and U (instead of T) as bases,
- is generally single stranded, though RNA molecules may have
hair-pins and double-stranded regions (even so often have imperfect
base-pairing).
Three types of RNA:
- Ribosomal (rRNA), which serves as a major component of the
machinery of protein biosynthesis.
- Transfer (tRNA), which serves as a "mechanical"
translation device to convert the four letter nucleic acid code
to the 20 letter amino acid code.
- Messenger (mRNA), which contains the primary sequence information
for the protein - acts as an information transfer tape.
All of the RNAs are complementary to a DNA strand, so the active
DNA (the strand which is "read") doesn't code, is the
non-sense strand. The sense strand of DNA, the strand which may
be directly out as a peptide sequence, is thus present only to
maintain the information integrity.
Look at mRNA and its transcription first (the rewriting of
the DNA information in slightly modified RNA format). mRNA is
used in protein biosynthesis, so look at protein expression first.
The proteins in a cell may be considered to be either of two types:
- Constitutive (house-keeping). These proteins are synthesized
at a constant rate, that is their expression is not dependent
on the environment - they are always present.
- Inducible. These proteins are synthesized in response to
the environment.
Look at E. coli lac operon as an example. [overhead]
There are significant differences between the RNA polymerase
systems of prokaryotes and eukaryotes. In particular the eukaryotic
systems studied have different polymerases for the different RNA
types, in addition to polymerases in mitochondria and chloroplasts,
while a single polymerase serves for E. coli. We will look
at the prokaryotic system first as the classic model, then come
back to the eukaryotic system.
E. coli RNA Polymerase
RNA polymerase requirements:
- Template - prefers double stranded DNA, but can use single
stranded.
- All four NTP's (as opposed to deoxyNTP's for DNA pol) - ATP,
CTP, GTP and UTP (note that U substitutes for T).
- Mg2+
Note that there is no primer requirement.
The reaction proceeds 5'Æ3',
with an attack of the 3'OH of the growing chain on the 5'-a-P of a free trinucleotide:
Note that this reaction will leave a triphosphate on the 5'-end
of a new RNA molecule.
RNA polymerase structure. The functional enzyme exists
in two forms:
- The Holoenzyme consisting of an a2bb's pentimer of 480 kD molecular weight,
making it one of the largest known soluble enzymes, with a diameter
of about 100 Å diameter. The holoenzyme is used to initiate
RNA biosynthesis, after which the sigma subunit dissociates to
give the core enzyme.
- The Core enzyme consisting of an a2bb' tetramer. The core enzyme synthesizes
RNA.
There are three stages in RNA polymerization:
- Initiation,
- Elongation,
- Termination.
Initiation: RNA polymerase binds to specific promoter
base sequences of about 40 nucleotides on the 5' side of the start
site, n = +1. (Note that sequences are labeled as -n...-1, +1...+n
where the (-) indicates the 5' side and (+) indicates the 3' side,
and there is no 0.)
- The core enzyme binds tightly, but non-specifically, to duplex
DNA. Addition of the sigma subunit gives the holoenzyme, which
binds only loosely to duplex DNA, allowing the the enzyme to
diffuse along the DNA strand until it reaches a promotor region.
The holoenzyme then binds tightly a region from about -20 to
+20. Within this region is a consensus sequence around -10, referred
to as a Pribnow box (= TATAAT, where TA ...T is highly conserved).
Additional less conserved sequences occur at about - 35 and +
5-8. These vary the efficiency of transcription (dependent on
the formation of an efficient complex).
- The holoenzyme binding to the promotor region results in
a closed complex. This complex isomerizes, causing promotor to
"melt", forming a "bubble", from -9 to +2
(the AT-rich sequence which has a lower binding energy, with
its fewer H-bonds, than a mixed or GC rich sequence, making this
opening easier).
- The new RNA sequence generally starts with a purine, usually
A, giving a 5' triphosphate: pppA + pppN Æ
pppApN + PPi. The polymerase synthesizes a
"primer" of about 10 nucleotides which forms one turn
of double helix with the DNA template, and the sigma unit is
released. At this point the enzyme changes from initiation to
elongation mode.
Elongation: The initiated RNA strand now grows 5'Æ3'.
- During elongation the RNA polymerase core enzyme contacts
about 30 nucleotides of DNA , of which perhaps 18 DNA base-pairs
are unwound (opened), and about 11 are base-paired to the new
RNA strand (approximately one turn of the helix).
- As synthesis proceeds we immediately run into a problem.
The DNA is a coil, so either the polymerase must run around the
coil, or the DNA must spin if it is left intact. Neither is likely
since both molecules are massive and embedded in a high viscosity
medium. To overcome this situation, topoisomerases are needed,
one before the transcription bubble to relax the supercoiling
as the bubble forms, and one behind to relax the supercoilling
the other direction as the helix reforms. The two nicks will
also allow the DNA bubble region to "spin" and the
RNA will thus not have to wrap around the DNA helix.
Last modified 26 March 2002
Pathway Diagrams
