Bacterial Transcription

Dr Mike Dyall-Smith, lab 3.07

Aim:

Understand the general process of bacterial transcription

References: Schaecter et al, Microbes, p141-8

Bacterial TranscriptionMain topics:

a) Overall scheme of information processing in cell

DNA RNA Protein (‘central dogma’)➔ ➔Transcription and Translation

b) Components of the transcription system in bacteria

RNA polymerase

DNA template, nucleotides, addition of new bases

c) Stages of the transcription process• RNAP binding to promoter, DNA unwinding, Initiation,

elongation, termination• Consensus promoters, Terminators

Main Points:

a) Overall scheme of information processing in cell

DNA → RNA → Protein (‘central dogma’)

Oscar Miller

Bacterial Transcription

DNA → RNA → Protein (‘central dogma’)

Bacterial Transcription

In prokaryotes, transcription and translation are directly connected

DNA

Transcription is the synthesis of an RNA molecule, called a transcript, from a DNA template.

Bacteria have only one RNA-P (eukarya have 3)

The bacterial RNA-P enzyme synthesises all the RNA species in the cell

Stable RNAs are tRNA, rRNA

Unstable RNA is mRNA, < 1min 1/2-life

Bacterial Transcription

Analysing transcriptsAnalysing transcriptsby Northern Blot hybridisation by Northern Blot hybridisation

Viral transcripts (RNA) separated by agarose gel electrophoresis.

Time post infection

Size of RNA

Viral transcripts (RNA) separated by agarose gel electrophoresis.

Time post infection

Size of RNA

RNA transferred to a membrane, hybridised to a labeled DNA probe to detect viral transcripts

Analysing transcriptsAnalysing transcriptsby Northern Blot hybridisation by Northern Blot hybridisation

Bacterial TranscriptionRNA polymerase (RNA-P):

Links ribonucleoside triphosphates (ATP, GTP, CTP and UTP) in 5’ - 3’ direction

Copies the DNA coding strand using the template strand

Can be modified to selectively transcribe genes by associating with sigma factors

Fig 14.6, Genes V (Lewin)

Phosphodiester bond formation

3’ end

Bacterial Transcription

• Note deoxythymidine in DNA is replaced by uridine in RNA

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Oscar Miller

RNA polymerase on virus promoters

E.coli RNAP, ~100 x 100 x 160 Å Darst et al., 1989

3D structure (from EM)

E.coli RNA polymerase

β

β'

ααω

σ

E.coli RNA polymerase

Darst et al., 1989

Core enzyme - will bind to any DNA at low affinity. Selective binding requires the activity of sigma factor.

sigma factor

omega factor - function unknown until recently.

β

β'

ααω

σ

α - 36.5 kDa, enzyme assembly, interacts with regulatory proteins, polymerisation

β' - 155 kDa, binds to DNA templateβ - 151 kDa, RNA polymerisation; chain initiation and

elongationσ - 70 kDa, promoter recognitionω - 11kDa, enzyme stability - restores denatured enzyme

E.coli RNA polymerase

Darst et al., 1989

β

β'

ααω

σ

E.coli RNAP - Sigma Factor

Sigma factor - • allows RNA pol to recognize promoters • reduces affinity to non-specific sites.

Specific for particular promoter sequences

Several different sigma factors for global controlσ70 is the basal sigma factor in E.coliσ32 is used after heat-shock

DNADNA ➙ RNARNA ➙ PROTEINPROTEINtranscription translation

Bacterial Transcription

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

• Transcription occurs at ~ 40 nucleotides/second at 37 ｡C (E.coli RNA pol.)

• Translation is ~ 15 amino acids/sec • Both are much slower than DNA replication

(800 bp/sec)

A Transcription Unit

DNA sequence transcribed into an RNA,

from promoter to terminator

Fig 14.6, Genes V (Lewin)

Binding

Initiation

elongation

Termination

Release

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

Terms1. TRANSCRIPTION: synthesis of RNA using a

DNA template

2.CODING STRAND: the DNA strand that is copied byRNA polymerase

3.TEMPLATE STRAND: the DNA strand used by RNA polymerase as the template. It is complementary to the coding strand, and the transcript.

4. TRANSCRIPT: the product of transcription.

Terms1. PROMOTER: The sequence of DNA needed

for RNA polymerase to bind and to initiate transcription.

2. START POINT: First base pair transcribed into RNA

3. UPSTREAM: sequence before the start point

4. DOWNSTREAM: sequence after the start point.

5. TERMINATOR: a DNA sequence that causes RNA pol to terminate transcription

Fig 14.14, Genes V (Lewin)

Typical Bacterial Typical Bacterial PromoterPromoter Sequence Sequence

Three main parts, the -35, -10 consensus sequences, and the start point.

Promoter for σ70 sigma factor of E.coli

PROMOTER - RNAPPROMOTER - RNAPone face of the DNA contacts the polymeraseone face of the DNA contacts the polymerase

Fig 14.16, Genes V (Lewin)

E.coliE.coli Sigma Factors Sigma Factors

Fig 14.16, Genes V (Lewin)

A Transcription Unit

DNA sequence transcribed into an RNA,

from promoter to terminator

Fig 14.6, Genes V (Lewin)

Binding

Initiation

elongation

Termination

Release

RNA being synthesised

Bubble

RNA pol activities

From www.ergito.com website

Diagram

From www.ergito.com website

Model

Yeast RNA pol

Model

Fig 14.11, Genes V (Lewin)

RNA Pol: Core and Holo enzyme are RNA Pol: Core and Holo enzyme are mainly found on DNA.mainly found on DNA.

500-1000 cRNAP at loose complexes

500-1000 hRNAP at loose complexes

Small % free hRNAP

500-1000 hRNAP in closed (or open) complexes at promoters

~ 2500 cRNAP actively transcribing genes.

Fig 14.12, Genes V (Lewin)

RNA Pol: finding promoters quicklyRNA Pol: finding promoters quickly

3 models

1. Random diffusion to target

2. Random diffusion to any DNA, followed by random displacement to any DNA

3. Sliding along DNA

Fig 14.12, Genes V (Lewin)

1. Random diffusion to target

2. Random diffusion to any DNA, followed by random displacement to any DNA

3. Sliding along DNA

3 models

Too slow

Unknown

Favoured

RNA Pol: finding promoters quicklyRNA Pol: finding promoters quickly

?

Fig 14.8, Genes V (Lewin)

Initial contact -55 to +20 = ~ 75 bp

RNA Pol binding to a promoter

Transcription occurs inside a region of opened DNA, a ‘bubble’.

The DNA duplex is unwound ahead of transcription, and reforms afterwards, displacing the RNA

Fig 14.3, Genes V (Lewin)

RNA Pol covers less DNA as it progresses from initiation to elongation. Partly because of sigma factor release, and partly from conformational changes of the core enzyme itself.

Fig 14.9, Genes V (Lewin)

Fig 14.15, Genes V (Lewin)

Footprint Analysis

Fig 14.15, Genes V (Lewin)

Footprint AnalysisRNAP+ RNAP-

Transcription Unit

DNA sequence transcribed into an RNA,

from promoter to terminator

Fig 14.14, Genes V (Lewin)

Transcription termination: Transcription termination: Intrinsic terminatorIntrinsic terminator

• stem-loop structures, 7-20 bp.

• GC-rich region followed by a poly-U region

• Structure forms within transcription bubble, making RNA-P pause

• A-U base pairs easily broken, leading to release of transcript

Fig 16.3, Genes V (Lewin)

Transcription termination: Transcription termination: Rho dependent terminatorRho dependent terminator

C-rich, G-poor region in RNApreceding termination

Fig 16.4, Genes V (Lewin)

Rho protein binds to RNA

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

Commercial transcription systemsCommercial transcription systems

Fig 16.4, Genes V (Lewin)

Phage RNA polymerases (T3, T7, SP6)

QuickTime™ and aSorenson Video decompressorare needed to see this picture.

Transcription - the movieTranscription - the movie

Watching single RNA polymerase enzymes move along a DNA template

RNAP attached to a plastic bead.

DNA (10kb) is attached at one end to a plastic bead, and tethered to a glass capillary.

Summary of Bacterial Transcription

Know the main terms in this process

Understand the process:

Template recognition: RNAP binds dsDNA

DNA unwinding at promoter

Initiation (short chains, 2-9nt, made)

Elongation (RNA made)

Termination (RNAP and RNA released)

Next lecture on regulation of gene expression

Transcription stages

Fig 14.6, Genes V (Lewin)