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Transcript
Transcription lecture notes
Overview
Synthesis of RNA shares many properties with the synthesis of DNA: both nucleic acids
are polymerized 5’3’ (with respect to strand being polymerized), from a single-stranded
template that is decoded 3’5’. rNTPs are the building blocks for RNA synthesis, and the
enzyme that carries out the reaction is RNA polymerase (RNA pol). The template for RNA
synthesis is DNA.
In bacteria, frequently have groups of genes that are transcribed together on single
mRNA (a polygenic or polycistronic mRNA. Such groups of genes called operons. An operon
will be transcribe from a single promoter (a DNA sequence that denotes a transcription start site).
Cellular content of RNA, DNA, and protein
The synthesis of RNA is an important cellular activity. A bacterial cell contains the following
amounts of these macromolecules, by percentage of cell dry weight (Table 3.2):
 16.4% rRNA
 2.5% tRNA
 1.6% mRNA
 3% DNA
 55% protein
Bacterial RNA polymerase
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Can initiate polymerization with 2 rNTPs
Must recognize where to start transcription, as RNA pols do not copy all DNA
RNA pol in Bacteria has a strongly conserved structure: 2’ core complex as the
primary catalytic unit. This enzyme is responsible for synthesis of all bacterial RNA
(except primers for DNA synthesis), in contrast to multiple dedicated enzymes, which
synthesize discrete RNA species in Eukaryotes. (The core RNA pol in at least many
bacteria also contains an omega subunit, but omega is not involved in RNA
polymerization).
A single RNA pol found in archeae, but the enzyme has a more complex structure: 8-10
subunits, more similar in its sequence to eukaryotic RNA pol
Enzyme in Bacteria has 2 states: Core RNA pol with 2’ and Holoenzyme with 2’
and a  subunit. Several different  subunits in the cell (see Table 7.3), which all serve
similar function, to help core RNA pol specifically recognize promoters.
Stages in transcription (Fig 7.21)
Initiation
RNA pol holoenzyme binding to dsDNA at promoter (e.g., Fig. 7.22); 12-18 bp are
unwound. First two rNTPs are joined; approximately 90% of time, first base is purine. Short
RNA is polymerized, then  subunit is released from RNA pol (leaving core to continue with
RNA elongation).
Elongation
Subsequent NTPs are added to growing RNA chain. RNA pol (core) moves down DNA
and unwinds another region of 12 bp DNA. Within transcription bubble, RNA/DNA
heteroduplex. Transcription continues at approximately 20 nt/sec, with RNA pol, pausing
frequently.
Termination
Occurs at specific sequences, either at intrinsic terminators (Fig. 7.23), or at sequences
dependent on protein called Rho (rho-dependent terminators. RNA/DNA heteroduplex falls
apart, and RNA pol falls off DNA.
Sequence elements of bacterial promoters (Fig. 7.22)
Promoter is dsDNA sequence, but usually described in terms of the sequence of sense
DNA strand (corresponds to the same sequence as RNA transcribed)
Promoter with 4 critical elements:
 -35 seqeuence
 -10 sequence (also called Pribnow box)
 spacing between –35 and –10 sequence
 RNA start site
Different  subunit-containing RNA pol holoenzymes will recognize promoters with
different sequences