L1 RNA-syntes Och Processning_HT11_Bilda

1L1: RNA Synthesis and Processing

Outline

Evolution of the gene concept

Transcription in Prokaryotes

Eukaryotic RNA Polymerases and General

Transcription Factors

Regulation of Transcription in Eukaryotes

RNA Processing and Turnover

Gene expression

Changes in gene expression, rather than losses of genetic

material, play a key role in guiding and maintaining cell

differentiation.

Gene expression changes regulate cell behaviours

including cell fate decisions during embryogenesis, cell

function, and chemotaxis, and can even lead to changes

in an entire organism, such as molting in insects

Gene expression is regulated by cell-extrinsic and cell-

intrinsic factors

What was a gene in da old days?

1860s-1900s: a discrete unit of heredity

1910s: a distinct locus

1940s: a blueprint for a protein

1960s: a transcribed code

1970s-1980s: an open reading frame (ORF) pattern

What was a gene yesterday (1990s-2000s)?

A DNA segment that contributes to phenotype/function. Inthe absence of demonstrated function a gene may becharacterized by sequencee, transcription or homology Wain et al, Genomics, 2002

A locatable region of genomic sequence, which isassociated with regulatory regions, transcribed regionsand/or other functional sequence regions Pearson,Nature, 2006

Alternatively spliced transcripts all belong to the same gene,even if the proteins that are produced are different theGene Sweepstake team, 2003

2The encode project-home work!

Nature 2007 Jun 14;447(7146):799-816

Some ENCODE findings

The human genome is pervasively transcribed, such that themajority of bases are associated with at least one primarytranscript, and many transcripts link distal regions toestablished protein coding loci.

Many novel non-protein coding transcripts have been identified,many of which overlap protein-coding loci and others locatedin regions previously thought to be silent

Numerous previously unrecognized transcript start sites havebeen identified.

Biological complexity revealed by ENCODE

Gerstein M B et al. Genome Res. 2007;17:669-6812007 by Cold Spring Harbor Laboratory Press

What is a gene today (post ENCODE project)?

A gene is a genomic sequence (DNA or RNA) directly encodingfunctional product molecules, either RNA or protein

In the case that there are several functional products sharingoverlapping regions, one takes the union of all overlappinggenomic sequences coding for them

This union must be coherent i.e., done separately for finalprotein and RNA products but does not require that allproducts necessarily share a common subsequence

or just

The gene is a union of genomic sequences encoding acoherent set of potentially overlapping functionalproducts

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3How the proposed definition of the gene can be applied to a sample case

Gerstein M B et al. Genome Res. 2007;17:669-681

2007 by Cold Spring Harbor Laboratory Press

The encode project-home work!

Genome Res. 2007 17: 669-681

What is a gene, post-ENCODE?History and updated definitionMark B. Gerstein, Can Bruce, Joel S. Rozowsky, Deyou Zheng, Jiang Du,Jan O. Korbel, Olof Emanuelsson, Zhengdong D. Zhang, Sherman Weissman,and Michael Snyder

While sequencing of the human genome surprised us with how many protein-coding genes there are, it did notfundamentally change our perspective on what a gene is. In contrast, the complex patterns of dispersed regulationand pervasive transcription uncovered by the ENCODE project, together with non-genic conservation and theabundance of noncoding RNA genes, have challenged the notion of the gene. To illustrate this, we review theevolution of operational definitions of a gene over the past centuryfrom the abstract elements of heredity ofMendel and Morgan to the present-day ORFs enumerated in the sequence databanks. We then summarize thecurrent ENCODE findings and provide a computational metaphor for the complexity. Finally, we propose a tentativeupdate to the definition of a gene: A gene is a union of genomic sequences encoding a coherent set of potentiallyoverlapping functional products. Our definition sidesteps the complexities of regulation and transcription byremoving the former altogether from the definition and arguing that final, functional gene products (rather thanintermediate transcripts) should be used to group together entities associated with a single gene. It also manifestshow integral the concept of biological function is in defining genes.

What is a gene?

gene

structural gene

RBS

promotorers

transcription start ATGGTG

TTG

TAA

TAG

TGA

signal peptide

transcription unit

transcription termination signal

Transcription in prokaryotes

1. Initiation: RNA-polymerase binds to the

promoter, conformational changes in the

promoter-polymerase complex (a bubble), initial

transcription

2. Elongation: conformational changes (tightened

grip around the template), unwinding of DNA,

RNA-synthesis, proof-reading

3. Termination: Rho-independent terminators

(intrinsic terminators = hairpin structures) or Rho-

dependent terminators

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4E. coli RNA polymerase

RNA polymerase: 2+ (ca 465 kDa)

- sigma factor influences the DNA binding

properties of the polymerase

- the affinity to different promoters can differ by

a factor of 106

- transcription is initiated w/o primer

Sequences of E. coli promoters

16-19 bp 5-9 bp

Start signals for RNA synthesis

Strong promoter: high affinity to RNA polymerase

Constitutive promoter: always on

Inducible promoter: possible to turn on and off at will

Sigma factors () in E. coli

stress responseSrpoS

heat shockErpoE

GCCGATAA15 bpCTAAAmotility/chemotaxis28 (F)fliA

TTGCA6 bpCTGGNAN-metabolism54rpoN

CCCGATNT13-15 bpCCCTTGAAheat shock32rpoH

TATAAT16-19 bpTTGACAgeneral70rpoD

-10sep-35UseFactorGene

Transcription by E. coli RNA polymerase (Part 1)

~ 12-14

bp

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5Transcription by E. coli RNA polymerase (Part 2)

(after ca 10 bp)

Transcription termination in Prokaryotes

RNA synthesis continues until the polymeraseencounters a termination signal.

The most common signal is a symmetricalinverted repeat of a GC-rich sequence followedby seven A residues.

Alternatively, transcription of some genes isterminated by a specific termination protein(Rho), which binds extended segments ofsingle-stranded RNA.

Transcription termination Transcription termination (E. coli)

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5

DNA

RNA

Promoter RNA polymerase

Hairpin termination signal

Other mechanism:

Termination via Rho protein (hexamer)

Rho

Pause-site

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6Transcription regulation

Two modes: positive and negative regulation

Positive regulation: transcription factors (activator/s) must bind

to the promoter in order for transcription to be initiated

- they help the RNA polymerase to bind the DNA (recruitment)

- allosteric influence on processes after the polymerase has

bound

Negative regulation: repressor protein binds to the operator

and prevents transcription

- hinders the RNA polymerase from binding the promoter

- alternatively, the RNA polymerase is retained at the promoter

Gene regulation after transcription initiation

Premature transcription termination, attenuation; e.g., trp

Anti termination = a type of positive transcription regulation in

which trancription factors (proteins) bind RNA polymerase and

modifies it so that it can read through special termination sites

- used by phages and in certain operons

Metabolism of lactose Negative control of the lac operon

7Positive control of the lac operon by glucose Regulation of the lactose operon (E. coli)

Lac Z Lac Y Lac A

Lac Z Lac Y Lac AS.D. S.D. S.D.

5 3

mRNA

DNA

-galactosidase:

Turns lactose into

galactose and glucose

Lactose permease:

Regulates the lactose

uptake

Thio-galactoside-acetylase:

Degrades non-cleavable

lactose analogues

mRNA

5 3Lac I

Lac I-35 -10-35 -10

Catabolite Activator

Protein (CAP)

Cyclic AMP

(cAMP)

cAMP/CAP-binding

region

mRNA-start

In the absence of lactose: Lac-repressor binds to the operator sequence Transcriptionen is blocked; no -galactosidase (or any of the other enzymers) is producced

Lactose (or IPTG) present: Lac-repressor cannot bind to the operator sequence

Transcription is allowed; -galactosidase (and all the other enzymes) is produced

Presence of lactose (or IPTG) and low concentrations of glucose: cAMP concentration rises

cAMP-CAP-complex formed that can bind upstream of the promoter

cAMP-CAP-complex promotes transcription (guides the RNA polymerase)

More -galactosidase is produced

Operator-sequence

Lac-repressor(Lac I)

Lac I

CAP

Promoter=Landing spot for

RNA polymerase

Lactose orIPTG

(synthetic analogue)

S.D.

RNA-pol.

Promoter

Eukaryotic RNA Polymerases and General Transcription Factors

Eukaryotic cells have three nuclear RNA

polymerases that transcribe different

classes of genes.

They are complex enzymes, consisting of

12 to 17 different subunits each.

They all have 9 conserved subunits, 5 of

which are related to subunits of bacterial

RNA polymerase.

Eukaryotic RNA Polymerases and General Transcription Factors

RNA polymerase II is responsible for synthesis of mRNA and ithas been the focus of most transcription studies.

Unlike prokaryotic RNA polymerase, it requires initiation factorsthat (in contrast to bacterial factors) are not associated withthe polymerase.

General transcription factors are proteins involved intranscription from all polymerase II promoters.

About 10% of the genes in the human genome encodetranscription factors, emphasizing the importance of theseproteins.

Promoters contain several different regulatory sequenceelements.

Promoters of different genes contain different combinations ofpromoter elements, which appear to function together to bindgeneral transcription factors.

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8Transcription Factors & Transcription Control in Eukaryotic Cells

How did eukaryotic organisms become so much morecomplex than prokaryotic ones, without a whole lotmore genes?

The answer lies in transcription factors (TFs)!

TFs recognizes specific DNA sequences and usually havemultiple functional domains for binding to e.g. TFs,coactivators, RNA pol II, chromatin remodelingcomplexes, ncRNAs etc.

The complexity and fine gradation of DNA expression ineukaryotes result from combinatorics; the combination ofchromatin and TF signals rather than the individual TFsignal is read out.

Transcription Factors & Transcription Control in Eukaryotic Cells

Does more cellular complexity require more genes?

Not really! E.g., it is estimated that humans haveapproximately one billion different kinds of neurons in thebrain while C. elegans (a roundworm) only has a total of 302neurons despite having nearly as many genes as we do.

The key to cell differentiation lies in the combination oftranscription factors (TFs) and chromatin structuresduring specific points of transition

Formation of a polymerase II preinitiation complex in vitro (Part 1) Formation of a polymerase II preinitiation complex in vitro (Part 2)

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9RNA polymerase II/Mediator complexes and transcription initiation The ribosomal RNA gene is transcribed by RNA polymerase I

Initiation of rDNA transcription Transcription of RNA polymerase III genes

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A eukaryotic promoter The SV40 enhancer

Action of enhancers DNA looping

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The immunoglobulin enhancer Structure of transcriptional activators

Examples of DNA-binding domains Action of transcriptional activators

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Action of eukaryotic repressors Regulation of transcriptional elongation (Part 1)

Regulation of transcriptional elongation (Part 2) Regulation of transcriptional elongation (Part 3)

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The packaging of eukaryoticDNA in chromatin hasimportant consequences fortranscription, so chromatinstructure is a critical aspect ofgene expression.

Modifications of chromatinstructure play key roles in thecontrol of transcription ineukaryotic cells (thought tohelp lock in expressionchanges needed duringdevelopment).

Actively transcribed genes arein relatively decondensedchromatin, which can be seenin polytene chromosomes ofDrosophila.


Chromatin can be modified by:

Interactions with HMG (high mobility

group) proteins

Modifications of histones

Rearrangements of nucleosomes

The modifications have either silencing or promoting

effects on gene expression

Histone acetylation (Part 1)

Histone modification:

The amino-terminal tail domains of corehistones are rich in lysine and can bemodified by acetylation (promotestranscription).

Transcriptional activators and repressorsare associated with histoneacetyltransferases (HAT) anddeacetylases (HDAC), respectively.

Patterns of histone modification

Can increase or decrase histone acetylation, thus having positive

and negative effetct on transcription, respectively

Promotes

transcription

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Chromatin remodeling factors

Chromatin remodeling factors are protein complexes that alter contacts between DNA

and histones.

They can reposition nucleosomes, change the conformation of nucleosomes, or eject

nucleosomes from the DNA.


To facilitate elongation, elongation factors become

associated with the phosphorylated C-terminal domain of

RNA polymerase II.

They include histone modifying enzymes and chromatin

remodeling factors that transiently displace nucleosomes

during transcription.

Regulation of Transcription in Eukaryotes: DNA methylation

DNA methylation is another general mechanism that controls

transcription in eukaryotes (gene silencing).

Methyl groups are added at the 5-carbon position of cytosines (C)

that precede guanines (G) (CpG dinucleotides).


Most newly-synthesized RNAs must be modified, except

bacterial RNAs which are used immediately for protein

synthesis while still being transcribed.

rRNAs and tRNAs must be processed in both prokaryotic

and eukaryotic cells.

Regulation of processing steps provides another level of

control of gene expression.

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Processing of ribosomal RNAs Processing of transfer RNAs (Part 1)

Processing of transfer RNAs (Part 2) Processing of eukaryotic messenger RNAs (Part 1)

(7-methylguanosine)

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Processing of eukaryotic messenger RNAs (Part 2) Formation of the 3 ends of eukaryotic mRNAs

RNA Processing and Turnover: Splicing

Three sequence elements of pre-mRNAs areimportant:- sequences at the 5 splice site, at the 3 splice site,and within the intron at the branch point.

Pre-mRNAs contain similar consensus sequences ateach of these positions.

Splicing takes place in large complexes, calledspliceosomes, which have five types of smallnuclear RNAs (snRNAs)U1, U2, U4, U5, and U6.

They are complexed with six to ten protein moleculesto form small nuclear ribonucleoprotein particles(snRNPs).

Splicing of pre-mRNA

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Alternative splicing in Drosophila sex determination Alternative splicing of Dscam

12x48x33x2=38016 combinations!!

RNA Processing and Turnover: RNA editing

RNA editing is processing (other than splicing) thatalters the protein-coding sequences of somemRNAs.

It involves single base modification reactions suchas deamination of cytosine to uridine andadenosine to inosine.

Editing of the mRNA for apolipoprotein B, whichtransports lipids in the blood, results in twodifferent proteins:

Apo-B100 is synthesized in the liver bytranslation of the unedited mRNA.

Apo-B48 is synthesized in the intestine fromedited mRNA in which a C has been changedto a U by deamination.

Editing of apolipoprotein B mRNA

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RNA Processing and Turnover: RNA degradation

Aberrant mRNAs can also be degraded.

Nonsense-mediated mRNA decay eliminatesmRNAs that lack complete open-reading frames.

When ribosomes encounter premature terminationcodons, translation stops and the defective mRNAis degraded.

Ultimately, RNAs are degraded in the cytoplasm.

Intracellular levels of any RNA are determined by abalance between synthesis and degradation.

Rate of degradation can thus control geneexpression.


rRNAs and tRNAs are very stable, in bothprokaryotes and eukaryotes.

Bacterial mRNAs are rapidly degraded, mosthave half-lives of 2 to 3 minutes.

In eukaryotic cells, mRNA half-lives vary; lessthan 30 minutes to 20 hours in mammalian cells.

Short-lived mRNAs code for regulatoryproteins, levels of which can vary rapidly inresponse to environmental stimuli.

mRNAs encoding structural proteins or centralmetabolic enzymes have long half-lives.


Degradation of eukaryote mRNAs is initiated byshortening of the poly-A tails.

Rapidly degraded mRNAs often contain specificAU-rich sequences near the 3 ends which arebinding sites for proteins that can eitherstabilize them or target them for degradation.

These RNA-binding proteins are regulated byextracellular signals, such as growth factors andhormones.

Degradation of some mRNAs is regulated by bothsiRNAs and miRNA.

mRNA degradation

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Summary

Transcription in prokaryotes and eukaryotes hassimilarities but is much more complex in eukaryotes

Many modes for regulation:- specificity factors- repressors/activators- general TFs- enhancers- chromatin structure- DNA methylation- post-transcriptional regulation

RNA processing is very important

Synthesis and turnover determines the final RNA levels;turnover is an important control mechanism

Identification of TFs & regulatory elements-home work!

Learn more about the following commonmethods for identification of TFs & regulatoryelements (use textbooks and/or internetbased info)

Transcription factor binding: DNA footprinting (Part 1) Transcription factor binding: DNA footprinting (Part 2)

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Transcription factor binding: Electrophoretic-mobility shift assay Transcription factor binding:

DNA footprinting

Gel shift assay

Identification of eukaryotic regulatory sequences Transcription factor binding: Chromatin immunoprecipitation (I)

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Transcription factor binding: Chromatin immunoprecipitation (II)

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L1 RNA-syntes Och Processning_HT11_Bilda