Transciption: RNA Polymerases and Transcription Event

Introduction

RNA Polymerases and the Transcription Event

Transcription Products

Introns, Exons, and Splicing hn RNA

Alternate Splicing of Exons

Course Topics

Course Home
Page

RNA Polymerases and Transcription Event

Bacterial RNA Polymerase

protein is about 480 kd in size
four subunits: alpha, beta, beta' and sigma; only the first three subunits are required for polymerase activity and are considered the core enzyme
the sigma factor is required for RNA polymerase to bind to the promoter; the enzyme has a loose affinity for DNA but when the sigma factor is present it will bind only at a promoter

Eukaryotic RNA Polymerase

three types exist

Type of Polymerase Product Location

RNA Polymerase I rRNA nucleolus

RNA Polymerase II hnRNA nucleoplasm

RNA Polymerase III tRNA nucleoplasm
protein is greater than 500 kd in size
two large subunits; <10 small subunits; largest subunit is homologous to beta', second largest subunit is homologous to the beta subunit
many non-polymerase factors required for binding of the enzyme to DNA

RNA polymerase produces a transcription unit that extends from the promoter to the termination sequences. The gene is defined in reference to the start site - those sequences before the start site are called the upstream sequences, those after the start site are called downstream sequences. The immediate product is the primary transcript.

The promoter region contains important sequences that are required for RNA polymerase to bind. These sequences are similar in both prokaryotic and eukaryotic genes, but the locations are different.

Conserved Sequences in Promoters

Cell Type	Location	Sequence
Prokaryotic	-10 -35	TATAAT TTGACA
Eukaryotic	about -25 about -80	TATA (TATA box) CAAT (CAAT box)

As DNA unwinds during bacterial transcription, the transcription apparatus occupies different sites in the gene during different steps of transcription.

Transcription Step Distance Covered Site

Initial complex 75 bp -55 to +20

Start of elongation 60 bp -35 to +25

During movement 30 bp 10 to +20 at start but moves as this size

DNA Footprinting Technique

The RNA polymerase-promoter complex is partially digested by DNase I. The DNA is then labeled on one strand. The fragments are then broken using the Maxam-Gilbert sequencing reagents. A control is run which is treated identically except it consists of the same promoter DNA without the transcription complex attached. The fragmented DNA is then separated on a gel. That region of the promoter sequence in the control that is missing in the complexed DNA defines the region in which the DNA is bound.

Steps in Model Eukaryotic Transcription

The adenovirus late promoter requires four accessory factors and RNA Polymerase II to be added to the promoter in a defined manner for transcription to begin. This can be monitored by footprinting analysis to measure the size of the complex. The following table shows the order in which the the four transcription factors and RNA Polymerase II come together to form the protein that will actually transcribe the gene.

Order Factor Protected Fragment

1. TFIID -42 to -17 (binds TATA box)

2. TFIIA -80 to -17

3. TFIIB -80 to -17 and -10 to +10

4. RNA Polymerase II -80 to +15

5. TFIIE -80 to +30

For bacterial RNA polymerase, only the sigma factor is needed for the enzyme to recognize the promoter. The eukaryotic transcription factors may indeed act as sigma factors to allow the eukaryoitic RNA polymerase to recognize the promoter. Some of the factors may be specific to certain promoters so groups of factors like these would be expected to exist.