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KAROLINSKA INSTITUTETDepartment of Cell and Molecular Biology

Medical Nobel Institute

Piergiorgio Percipalle, PhD

Tutorial IITutorial IIGene expression:Gene expression:

Molecular mechanisms of gene Molecular mechanisms of gene transcriptiontranscription Control of gene transcription and RNA

processing

mRNA translation and protein synthesis

ProgramProgram

Gene transcriptionHow do cells use information storedIn the chromosomes?

Schematic representation of chromosome 2 from the fruit fly (D. Melanogaster)

The central dogma of molecular biology

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RNA synthesis

DNA sequences are transcribed to RNA

TranskriptionTranskriptionTranskription

Ribonucleic acid (RNA)

In the RNAmolecule, nucleotides are called”ribonucleotides”RNA contains the base ”uracil” instead of ”thymine”.Thymine can also base-pair with adenine

RNA is a linear polymer, just like DNA, consisting of4Different nucleotides linked to each other through phosphodiester bonds

Similarities between DNA and RNA

Differences between DNA and RNA

The chemical structure in theRNA molecule

AA

BB

Uracil base-pairs with adenine DNA have RNA have differentternary structures

DNA is a duplexRNA is linear

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Type of RNA Function

mRNAs

rRNAs

tRNAs

snRNAs

snoRNAs

Non-coding RNAs

messanger-RNA, protin-coding

ribosomal-RNAs, ribosome structure

transfer-RNAs, protein synthesis

small nuclear RNAs, RNA processing

small nuclear RNAs, rRNA processing

different cellular processes

There are several types of RNA

How is a specific RNA producedform a template DNA?

Transcription is performed byRNA polymerase

Recognition of a specific DNA sequence:RNA synthesis starts

RNA synthesis

Promoter

Terminator

Transcription is a step-wise process

Recognition of a specific DNA sequence:RNA synthesis ends

The transcription cycle

InitiationTermination

Elongation

RNA polymerase

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Direction of RNA polymerase Transcription along a chromosome

Type of polymerase Genes whichare transcribed

RNA polymerase I

RNA polymerase II

RNA polymerase III

rRNA genes

all protein-coding genes

tRNA genes

Eukaryotes have three differentRNA polymerases

The structure af RNA polymerase IIis very conserved

In eukaryotic cells, RNA polymerase II transcription requires regulatory

sequences on the DNA

UAS

RNA polymerase II requires generaltranscription factors……..

TranscriptionTranscription

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Factor No of subunits Function

TFIIA 2 Stabilizes TBP and TFIID DNA binding.Blocks transcription inhibitors. Positive andnegative gene regulation

TFIIB 1 Binds TBP, Pol II and promoter DNA. Helpsfix transcription start site

TFIID TBP 1 Binds TATA element and deforms promoter DNA.Platform for assemblyof TFIIB, TFIIA and TAFs.

TAFs 14 Binds INR and DPE promoter elements. Targetof regulatory factors.

TFIIF 3 Binds Pol II and is involved in Pol II recruitmentto PIC and in open complex formation.

TFIIE 2 Binds promoter near transcription start. Mayhelp open or stabilize the transcription bubblein the open complex.

TFIIH 10 Functions in transcription and DNA repair.Kinase and two helicase activities. Essential foropen complex formation. Mutations in IIH cancause disease.

RNA polymerase II generaltranscription factors

From Hahn, Nature Structural & Molecular Biology 2004

Mediator

• Predominant role to enhance the PIC assembly, via pol II

Formation of ribonucleoprotein(RNP) particles

Transport facilitatorTransport mediatorSplicing factorSplicing repressorTranslational regulator

DNA

Pre-mRNA

RNA polymerase II

Daneholt, Proc. Natl. Acad. Sci. USA 2001

Chromatin structure

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 transcriptionand chromatin?

Pre-mRNARNA polymerase IIC-terminal domain

RNA processing

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Formation of ribonucleoprotein(RNP) particles

Transport facilitatorTransport mediatorSplicing factorSplicing repressorTranslational regulator

DNA

Pre-mRNA

RNA polymerase II

Daneholt, Proc. Natl. Acad. Sci. USA 2001

In eukaryotes RNA elongationoccurs concomitantly with processing

RNA splicing

RNA polyadenylation (3’ end)

RNA capping (5’ end)

RNA-capping

Methylation of guanosine

5’ - phopsphate released

GMP addition(5’ to 5’)

RNA splicing

In eukaryotic cells, the primary RNA transcript(pre-mRNA), includes both coding andnon-coding sequences

In pre-mRNA, coding sequences are called Exonswhereas non-coding sequences are calledIntrones

Examples of gene structures During splicing intronic sequencesare removed

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RNA splicing is mediated by the Spliceosome

AA

BB

CC

DD

EE

FF

An example of alternative splicing:the α-tropomyosin gene

RNA polyadenylation

PAP adds 200 nucleotidesat the 3’ end of the RNA transcript

Termination sequences

RNA cleavage

The Cell Nucleus by Ramon y Cajal(Nobel Prize, Medicine and Physiology)

The structure of the cell nucleusThe structure of the cell nucleus The nucleolus and its subdomainsThe nucleolus and its subdomains

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Mechanisms of rRNA processing Visualization of nuclear structures:Cajal Bodies

Human cells

Xenopus Oocytes

FunctionFunction ofof Cajal BodiesCajal Bodies

Cajal Bodies/GEMSAssembly of Transcription-ProcessingMachinery

Cajal Cajal Bodies/GEMSBodies/GEMSAssemblyAssembly of of TranscriptionTranscription--ProcessingProcessingMachineryMachinery

Visualization of Speckles

Stockpiles of fully mature snRNPs Stockpiles of fully mature snRNPs ready to be used in RNAready to be used in RNA--splicingsplicing

Visualization of chromatin andVisualization of chromatin andnuclear structurenuclear structure

Cajal Body

Cajal Body

Speckles

Speckles

Nucleo cytoplasmic transport ofmature mRNA molecules

ProteinSynthesis

Protein synthesis factors