CBG.

04: Transcription

TRANSCRIPTION PRODUCES RNA RNA POLYMERASE transcribes genetic
COMPLEMENTARY TO ONE STRAND OF DNA information from DNA into RNA

- Transcription differs from DNA replication in - has many moving parts that form a machine that
several crucial ways: surrounds DNA strands, unwinds them, and builds
- unlike the newly formed DNA strand, the RNA an RNA strand based on the information held
strand does not remain hydrogen-bonded to the DNA inside the DNA
template strand; instead, just behind the region where the - once the enzyme gets started, RNA polymerase
ribonucleotides are being added, the RNA chain is displaced marches confidently along the DNA
and the DNA helix re-forms
- RNA molecules are much shorter than DNA
molecules, because they are copied from only a limited
region of the DNA
- RNA polymerase catalyse the formation of the
phosphodiester bonds that link the nucleotides together to
form a linear chain; the RNA chain is extended in the 53
direction
- The substrates are nucleoside triphosphates (ATP,
CTP, UTP, GTP)
- a hydrolysis of high-energy bonds provides the
energy needed to drive the reaction forward
- the synthesis of additional RNA molecules is
started before the first RNA is completed; when RNA
polymerase molecules follow hard on each others heels in
this way, each moving at about 20 nt/s (in EK), over a
thousand transcripts can be synthesised in 1hr from a
single gene
- RNA polymerase catalyzes the linkage of
ribonucleotides, not deoxyribonucleotides
- RNA polymerase can start an RNA chain without a
primer
- although RNA polymerases are not nearly as
accurate as the DNA polymerases that replicate DNA, they
have a proofreading mechanis: if the incorrect - the active site is designed to be able to remove
ribonucleotide is added to the growing RNA chain, the nucleotides as well as add them to the growing
polymerase can back up, and the active site of the enzyme strand; the enzyme tends to hover around
can perform an excision reaction that mimics the reverse of mismatched nucleotides longer than properly
the polymerisation reaction, except that water instead of added ones, giving the enzyme time to remove
pyrophosphate is used; them; this process is somewhat wasteful, since
- RNA polymerase hovers around a misincorporated proper nucleotides are also occasionally removed
ribonucleotide longer than it does for a correct addition, - it is a sensitive target for poisons and toxins:
causing excision to be favoured for incorrect nucleotides; alpha-amanitin, a small circular peptide created by
however, RNA polymerase also excises many correct bases the death cap mushroom; it binds to the back side
as part of the cost for improved accuracy of RNA polymerase, away from the active site and
away from the binding site for the DNA are RNA
Replication step Errors/ nucleotide
it does not physically block the active sire, but instead
5 3 polymerization 1 105 jams the mechanism of the enzyme; the poison binds
between the 2 subunits of the protein, gluing them
together and blocking the essential motions;
3 5 exonucleolytic 1 102
proofreadinf

Strand-directed mismatch 1 102
repair
Total 1 109


TATA-BINDING PROTEIN tells RNA
polymerase where to get started on a gene

- RNA polymerase unwinds the 2 strands of DNA
and transcribes the genetic information into a strand of
RNA
- in bacteria, typical promoters contain 2 regions
that interact with the sigma subunit of their RNA
polymerase; the sigma subunit binds to these DNA
sequences, assists the start of transcription, and then
detaches from the polymerase as it continues transcription
through the gene
- EK cells have a far more complex promoter
system, using dozens of different proteins to ensure that
the proper RNA polymerase is targeted to each gene; the
TATA-binding protein is the central element of this system
- The typical sequence is something like: T-A-T-A-
a/t-A-a/t; surprisingly, many variations on this theme also
work

- TBP binds to TATA sequence, creating a landmark
that marks the start site of transcription

- TATA-binding protein works as part of a larger - TBP uses 2 types of interactions to recognize and
transcription factor TFIID, that starts the process of hold the TATA sequence;
transcription; after it binds to the promoter, it recruits - It has a string of lysine and arginine AA that
additional transcription factors; TFIIB binds next; then, a interact with the phosphate groups of the DNA; this glues
string of other transcription factors bind, constructing a the protein to the DNA
large protein complex that decides whether or not to start - the protein uses specially-placed AA to interact
transcription; these may include transcription activators, with DNA bases;
such TFIIA, that promotes the start of transcription; - 4 phenylalanine AA jam into the DNA minor
- Other factors inhibit the start of transcription, groove and form the kinks that bend the DNA; there are 2
such as transcription regulator NC2 (negative cofactor 2) symmetrical asparagine that form H bonds at the very
centre;
- the combination of the unusual flexibility of TATA
DNA sequences and these specific hydrogen bonds allow
TBP to recognize the proper sequence
- composed of 2 symmetrical halves