Download Gene transcription

Tutorial II
Gene expression:
Molecular mechanisms of gene
transcription
Piergiorgio Percipalle, PhD
Program
Control of gene transcription and RNA
processing
mRNA translation and protein synthesis
KAROLINSKA INSTITUTET
Department of Cell and Molecular Biology
Medical Nobel Institute
Gene transcription
How do cells use information stored
In the chromosomes?
The central dogma of molecular biology
Schematic representation of
chromosome 2 from the fruit
fly (D. Melanogaster)
1
DNA sequences are transcribed to RNA
RNA synthesis
Ribonucleic acid (RNA)
Similarities between DNA and RNA
Transkription
Transkription
The chemical structure in the
RNA molecule
A
RNA is a linear polymer, just like DNA, consisting of4
Different nucleotides linked to each other through
phosphodiester bonds
Differences between DNA and RNA
B
In the RNAmolecule, nucleotides are called
”ribonucleotides”
RNA contains the base ”uracil” instead of ”thymine”.
Thymine can also base-pair with adenine
Uracil base-pairs with adenine
DNA have RNA have different
ternary structures
DNA is a duplex
RNA is linear
2
There are several types of RNA
Type of RNA
mRNAs
Function
messanger-RNA, protin-coding
rRNAs
ribosomal-RNAs, ribosome structure
tRNAs
transfer-RNAs, protein synthesis
snRNAs
small nuclear RNAs, RNA processing
snoRNAs
small nuclear RNAs, rRNA processing
Non-coding RNAs
different cellular processes
Transcription is performed by
RNA polymerase
How is a specific RNA produced
form a template DNA?
Transcription is a step-wise process
Recognition of a specific DNA sequence:
RNA synthesis starts
Promoter
RNA synthesis
Recognition of a specific DNA sequence:
RNA synthesis ends
The transcription cycle
Termination
Terminator
RNA polymerase
Initiation
Elongation
3
Direction of RNA polymerase
Eukaryotes have three different
RNA polymerases
Type of polymerase
Transcription along a chromosome
The structure af RNA polymerase II
is very conserved
Genes which
are transcribed
RNA polymerase I
rRNA genes
RNA polymerase II
all protein-coding genes
RNA polymerase III
tRNA genes
In eukaryotic cells, RNA polymerase II
transcription requires regulatory
sequences on the DNA
RNA polymerase II requires general
transcription factors……..
UAS
Transcription
4
RNA polymerase II general
transcription factors
Factor
No of subunits
TFIIA
2
TFIIB
TFIID
1
TBP
TAFs
1
14
TFIIF
3
TFIIE
2
TFIIH
10
Mediator
Function
Stabilizes TBP and TFIID DNA binding.
Blocks transcription inhibitors. Positive and
negative gene regulation
Binds TBP, Pol II and promoter DNA. Helps
fix transcription start site
Binds TATA element and deforms promoter DNA.
Platform for assemblyof TFIIB, TFIIA and TAFs.
Binds INR and DPE promoter elements. Target
of regulatory factors.
Binds Pol II and is involved in Pol II recruitment
to PIC and in open complex formation.
Binds promoter near transcription start. May
help open or stabilize the transcription bubble
in the open complex.
Functions in transcription and DNA repair.
Kinase and two helicase activities. Essential for
open complex formation. Mutations in IIH can
cause disease.
• Predominant role to enhance the PIC assembly, via
pol II
From Hahn, Nature Structural & Molecular Biology 2004
Formation of ribonucleoprotein
(RNP) particles
Chromatin structure
Pre-mRNA
DNA
RNA polymerase II
Transport facilitator
Transport mediator
Splicing factor
Splicing repressor
Translational regulator
Daneholt, Proc. Natl. Acad. Sci. USA 2001
Chromatin structure modifications
Chromatin and histone modifications
as part of relay mechanisms
Percipalle and Östlund Farrants, Curr Op Cell Biol 2006
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Summary of the transcription
initiation process in eukaryotes
(Saccharomyces cerevisiae)
Eukaryotic gene repressors
Repressors compete with activators
Crosstalk between transcription
and chromatin?
RNA processing
RNA polymerase II
C-terminal domain
Pre-mRNA
6
Formation of ribonucleoprotein
(RNP) particles
In eukaryotes RNA elongation
occurs concomitantly with processing
RNA capping (5’ end)
Pre-mRNA
RNA splicing
DNA
RNA polymerase II
RNA polyadenylation (3’ end)
Transport facilitator
Transport mediator
Splicing factor
Splicing repressor
Translational regulator
Daneholt, Proc. Natl. Acad. Sci. USA 2001
RNA-capping
RNA splicing
5’ - phopsphate released
GMP addition
(5’ to 5’)
Methylation of guanosine
Examples of gene structures
In eukaryotic cells, the primary RNA transcript
(pre-mRNA), includes both coding and
non-coding sequences
In pre-mRNA, coding sequences are called Exons
whereas non-coding sequences are called
Intrones
During splicing intronic sequences
are removed
7
RNA splicing is mediated by the
Spliceosome
A
B
E
C
F
An example of alternative splicing:
the α-tropomyosin gene
D
RNA polyadenylation
The Cell Nucleus by Ramon y Cajal
(Nobel Prize, Medicine and Physiology)
Termination sequences
RNA cleavage
PAP adds 200 nucleotides
at the 3’ end of the RNA transcript
The structure of the cell nucleus
The nucleolus and its subdomains
8
Mechanisms of rRNA processing
Visualization of nuclear structures:
Cajal Bodies
Human cells
Xenopus Oocytes
Function of Cajal Bodies
Visualization of Speckles
CajalBodies/GEMS
Bodies/GEMS
Cajal
Assemblyof
of
Assembly
Transcription-Processing
Transcription-Processing
Transcription
Machinery
Machinery
Stockpiles of fully mature snRNPs
ready to be used in RNARNA-splicing
Visualization of chromatin and
nuclear structure
Speckles
Cajal Body
Nucleo cytoplasmic transport of
mature mRNA molecules
Protein synthesis
factors
Protein
Synthesis
Cajal Body
Speckles
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