Transcript Streptococcus pyogenes

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 Transcription
Main 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
Bacterial Transcription
Main Points:
a) Overall scheme of information processing in cell
DNA → RNA → Protein (‘central dogma’)
Oscar Miller
Bacterial Transcription
DNA → RNA → Protein (‘central dogma’)
DNA
In prokaryotes, transcription and translation are directly connected
Bacterial Transcription
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
Analysing transcripts
by Northern Blot hybridisation
Viral transcripts (RNA)
separated by agarose gel
electrophoresis.
Size of RNA
Time post infection
Analysing transcripts
by Northern Blot hybridisation
Viral transcripts (RNA)
separated by agarose gel
electrophoresis.
Size of RNA
Time post infection
RNA transferred to a membrane, hybridised to a
labeled DNA probe to detect viral transcripts
Bacterial Transcription
RNA 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
Phosphodiester bond formation
3’ end
Fig 14.6, Genes V (Lewin)
Bacterial Transcription
•
Note deoxythymidine in DNA is replaced
by uridine in RNA
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
E.coli RNA polymerase
RNA polymerase on virus
promoters
Oscar Miller
3D structure (from EM)
E.coli RNAP, ~100 x 100 x 160 Å
Darst et al., 1989
E.coli RNA polymerase
omega factor function unknown until
recently.
Darst et al., 1989
σ
β'
α
ω
sigma factor
α
β
Core enzyme - will bind to any DNA at low affinity. Selective
binding requires the activity of sigma factor.
E.coli RNA polymerase
σ
β'
α
ω
Darst et al., 1989
α
β
α - 36.5 kDa, enzyme assembly, interacts with regulatory
proteins, polymerisation
β' - 155 kDa, binds to DNA template
β - 151 kDa, RNA polymerisation; chain initiation and
elongation
σ - 70 kDa, promoter recognition
ω - 11kDa, enzyme stability - restores denatured enzyme
E.coli RNAP - Sigma Factor
β'
α
ω
α
β
σ
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 control
σ70 is the basal sigma factor in E.coli
σ32 is used after heat-shock
Bacterial Transcription
DNA ➙ RNA
transcription
•
•
•
➙
PROTEIN
translation
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)
Qu i c k T i m e ™ a n d a
T I F F (L Z W ) d e c o m p re s s o r
a re n e e d e d t o s e e t h i s p i c tu re .
A Transcription Unit
Binding
DNA sequence
transcribed into an RNA,
from promoter to
terminator
Initiation
Release
Termination
elongation
Fig 14.6, Genes V (Lewin)
Qu i c k T i m e ™ a n d a
T I F F (L Z W ) d e c o m p re s s o r
a re n e e d e d to s e e th i s p i c t u re .
Terms
1. TRANSCRIPTION: synthesis of RNA using a DNA
template
2. CODING STRAND: the DNA strand that is copied by
RNA 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.
Terms
1.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
Typical Bacterial Promoter Sequence
Three main parts, the -35, -10 consensus sequences,
and the start point.
Promoter for σ70 sigma factor of E.coli
Fig 14.14, Genes V (Lewin)
PROMOTER - RNAP
one face of the DNA contacts the polymerase
Fig 14.16, Genes V (Lewin)
E.coli Sigma Factors
Fig 14.16, Genes V (Lewin)
A Transcription Unit
Binding
DNA sequence
transcribed into an RNA,
from promoter to
terminator
Initiation
Release
Termination
elongation
Fig 14.6, Genes V (Lewin)
RNA being
synthesised
RNA pol activities
Bubble
Diagram
From www.ergito.com website
Model
From www.ergito.com website
Model
Yeast RNA pol
RNA Pol: Core and Holo enzyme are
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 14.11, Genes V (Lewin)
RNA 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)
RNA Pol: finding promoters quickly
3 models
1. Random diffusion to target
Too slow
2. Random diffusion to any DNA,
followed by random displacement to
any DNA
Favoured
Unknown
Fig 14.12,
Genes V (Lewin)
?
3. Sliding along DNA
Initial contact -55 to +20 = ~ 75 bp
RNA Pol
binding to a
promoter
Fig 14.8, Genes V (Lewin)
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)
Footprint Analysis
Fig 14.15, Genes V (Lewin)
Footprint Analysis
RNAP+
Fig 14.15, Genes V (Lewin)
RNAP-
Transcription Unit
DNA sequence
transcribed into an RNA,
from promoter to
terminator
Fig 14.14, Genes V (Lewin)
Transcription termination:
Intrinsic terminator
• stem-loop structures, 7-20 bp.
• GC-rich region followed by a polyU 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:
Rho dependent terminator
Rho protein binds to RNA
C-rich, G-poor region in RNA
preceding termination
Fig 16.4, Genes V (Lewin)
Qu i c k T i m e ™ a n d a
T I F F (L Z W ) d e c o m p re s s o r
a re n e e d e d to s e e th i s p i c t u re .
Commercial transcription systems
Phage RNA polymerases (T3, T7, SP6)
Fig 16.4, Genes V (Lewin)
Transcription - the movie
DNA (10kb)
is attached at
one end to a
plastic bead,
and tethered
to a glass
capillary.
QuickTime™ and a
Sorenson Video decompressor
are needed to see this picture.
Watching single RNA polymerase
enzymes move along a DNA template
RNAP
attached to a
plastic bead.
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)