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)

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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

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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

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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

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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

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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

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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

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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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