Transcription: BWC
In bacteria:
Initiation:
using incoming ribonucleoside triphosphate molecules (NTPs) as substrates
first two incoming NTPs are hydrogen-bonded to the complementary bases of the DNA template
strand at the start site, RNA polymerase catalyzes the formation of a phosphodiester bond
between the 3′-hydroxyl group of the first NTP and the 5′-phosphate of the second, accompanied
by the release of pyrophosphate (PPi).
The polymerase then advances along the template strand as additional nucleotides are added one
by one, the 5′-phosphate of each new nucleotide joining to the 3′-hydroxyl group of the growing
RNA chain. Thus, unlike DNA polymerase (see Chapter 17), RNA does not need primers.
RNA polymerase initiates synthesis until the chain is nine nucleotides long or shorter
a piece of RNA ten nucleotides or longer is produced, and this weakens the interactions of RNA
polymerase with the promoter, allowing it to escape the promoter. Once it escapes, the RNA
polymerase releases sigma factor, allowing the rest of the polymerase molecule to move along the DNA
to transcribe an elongating RNA
Elongation:
The b subunits contain regions that act like “pincers” that bind DNA and move it into the active site of
the enzyme
Because complementary base pairing between the DNA template strand and the newly forming RNA
chain is antiparallel, the RNA strand is elongated in the 5′ S 3′ direction as each successive nucleotide is
added to the 3′ end of the growing chain.
most recently added nucleotides remain base-paired with the DNA template strand, forming a short
RNA-DNA hybrid about 8–9 bp long.
Proofreading: in 2 ways
1. When an incorrect ribonucleotide is put into the RNA, RNA polymerase waits at that position, making
it more likely that the reverse reaction will occur
2. When a noncomplementary nucleotide is incorporated into a growing RNA chain by mistake, the RNA
polymerase backs up slightly, transcription stalls, and the noncomplementary nucleotide participates in
catalyzing its own removal by RNA polymerase along with removal of the previously incorporated
nucleotide, a process called RNA backtracking.
Termination:
a termination signal, that triggers the end of transcription
In bacteria, two classes of termination signals can be distinguished based on whether they require the
participation of a protein called rho (R) factor.
RNA molecules terminated without the aid of the rho factor contain a short GC-rich sequence followed
by several U residues near their 3′ end
First, the GC region contains sequences that are complementary to each other, causing the RNA to
spontaneously fold into a hairpin loop that tends to pull the RNA molecule away from the DNA. Then the
weaker bonds between the sequence of U residues and the DNA template are broken, releasing the
newly formed RNA molecule.
Some genes were found to be transcribed into RNA molecules that are longer than the RNAs produced
in living cells, suggesting that transcription was not terminating properly. =>
This problem could be corrected by adding rho factor, which binds to specific termination sequences
50–90 bases long located near the 3′ end of newly forming RNA molecules.
The rho factor acts as an ATP-dependent unwinding enzyme, moving along the newly forming RNA
molecule toward its 3′ end and unwinding it from the DNA template as it proceeds.
In eukaryotes:
RNA polymerase I - resides in the nucleolus and is responsible for synthesizing an RNA molecule that
serves as a precursor for three of the four types of rRNA found in eukaryotic ribosomes (28S rRNA, 18S
rRNA, and 5.8S rRNA).
RNA polymerase II- is found in the nucleoplasm and synthesizes precursors to mRNA, the class
of RNA molecules that encode proteins. Rather than being diffusely distributed throughout the
nucleus, active molecules of polymerase II are located in discrete clusters, called transcription
factories, that may represent sites where active genes come together to be transcribed. In addition
to producing mRNA precursors, RNA polymerase II synthesizes most of the snRNAs, small
nuclear RNAs involved in posttranscriptional RNA processing, and the microRNAs, which
regulate the translation and stability of specific mRNAs and, to a lesser extent, control the
transcription of certain genes. Polymerase II is responsible for producing the greatest variety of
RNA molecules and is extremely sensitive to a-amanitin, which explains the toxicity of this
compound to humans and other animals.
RNA polymerase II differs from polymerases I and III at its C-terminus, where it has extra amino acids.
RNA polymerase III - it synthesizes a variety of small RNAs, including tRNA precursors and the smallest
type of ribosomal RNA, 5S rRNA. Mammalian RNA polymerase III is sensitive to a-amanitin but only at
higher levels of the toxin than are required to inhibit RNA polymerase II. (The comparable enzymes of
some other eukaryotes, such as insects and yeasts, are insensitive to a-amanitin.)
3 classes of promoters:
core promoter- defined as the smallest set of DNA sequences able to direct the accurate initiation of
transcription by RNA polymerase, actually extends into the nucleotide sequence to be transcribed
These four elements are (1) a short initiator (Inr) sequence surrounding the transcriptional start site
(which is often an A, as in bacteria); (2) the TATA box, which consists of a consensus sequence of TATA
followed by two or three more A’s, usually located about 25 nucleotides upstream from the start site;
(3) the TFIIB recognition element (BRE) located slightly upstream of the TATA box; and (4) the
downstream promoter element (DPE) located about 30 nucleotides downstream from the start site.
These four elements are organized into two general types of core promoters:
TATA-driven promoters, which contain an Inr sequence and a TATA box with or without an associated
BRE
DPE-driven promoters, which contain DPE and Inr sequences but no TATA box or BRE.
Besides being found in eukaryotes, TATA-driven promoters are also present in archaea, a key piece of
evidence supporting the idea that in some ways, archaea resemble eukaryotes more closely than they
resemble bacteria
core promoter (TATA-driven or DPE-driven) is capable of supporting only a basal (low) level of
transcription.
RNA polymerase III molecule uses promoters that are entirely downstream of the transcription unit’s
transcriptional start site when transcribing genes for tRNAs and 5S rRNA.
The tRNA promoter has consensus sequences called box A and box B. The promoters for 5S-rRNA genes
have box A (positioned farther from the start site than in tRNA-gene promoters) and another critical
sequence, called box C.