Transcription Lecture Notes

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

· 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: a2bb’ 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 a2bb’ and Holoenzyme with a2bb’ and a s subunit. Several different s 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 s 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 s subunit-containing RNA pol holoenzymes will recognize promoters with different sequences