Gene Expression

1: Activation (overview), Transcription, Translation.

The Goodwin Equations

Review An active gene is one that is being

transcribed, and whose transcription product is being translated, and whose translation product affects cellular behavior/development.

Genes are activated by transcription factors – the principal way transcription is controlled

Transcription factors are proteins that themselves are gene products.

Transcription factors often require a cofactor, a very important environmental signal.

Gene Activation is a control system

Transcription factor Active Gene

Transcription Product:

Messenger RNA

Translation Product:

(Active Protein)

Change in Change in

transcription factorcofactor level

activity

Thus, one has a rudimentary feedback loop

Since the transcription factor is itself a gene product the feedback loop can (and usually does) involve multiple genes. Since the cofactor level may involve environmental stimuli the feedback loop can involve their signal transduction pathways.

Gene Expression is best understood by mapping,

quantifying, and understandings the behavior of these loops.

Transcription

A good reference:

(Blackwell, 2001)

Genes are transcribed (copied) into 'messenger RNA' by an enzyme called

RNA Polymerase [RNAP].

Polymerase binds to a 'promoter region' at the beginning of a gene.

The polymerase traverses the gene, copying as it goes.

Polymerase normally leaves DNA at the gene's end.

Multiple polymerases may be attached to one gene at any given time.

The transcription

“bubble”, White, Fig.

1.8

Three kinds of RNAP

Pol I – rRNAPol II – mRNA and snRNA (small nuclear

RNA, involved in splicing)Pol III – small RNA’s (tRNA, 5s rRNA …)

We will concentrate on Pol II although many features are common to all three.

Model of Pol II RNAP (White)

What determines the rate of transcription?

Transcription velocity is mostly constant, over one gene and from gene to gene.

Transcription length is determined by the gene. Thus …

(Molar) synthesis rate for transcription is controlled by gene length, number of RNAP's on the gene.

Rates (Hargrove): 2500 nt min-1 (procaryotes), 3000 nt min -1 (eukaryotes)

The initiation rate for transcription*

is the most important factor determining which gene products

are generated

* i.e. the attachment and hence (in steady state) the detachment rate for RNA polymerase (RNAP). This determines the average number of mRNA's on the gene.

What determines the rate of translation (mRNA protein)?

Translation is effected by ribosomes, complex enzymes made of both protein and nucleic acid, that traverse mRNA's and translate their codons (RNA triplets) into amino acid sequences.

In a manner similar to transcription, the ribosome traverses at a near-constant velocity. Thus …

for translation …

(Molar) synthesis rate is controlled by ribosomal attachment rate, mRNA length, and the number of mRNA's present.

Rates (Hargrove): 2700 nt min-1 (procaryotes), 720 nt min -1 (eukaryotes)

Summary of Gene Expression Rates Typical values for parameters and rates needed to quantify gene expression in E. coli and mammalian

cells (from Hargrove)

(nt = nucleotides) E. coli MammalsInitiation 1 sec-1 10 min -1

Transcription 2500 nt min-1 3,000 nt min-1

RNA processing not applicable ca. 10 min

Half-life, nuclear RNA not applicable ca. 10 min

Nucleocytoplasmic transport

not applicable ca. 10 min

Half-life, mRNA 1-3 min 1-20 h

mRNA translation 2700 nt min-1 720 nt min-1

Half-life, protein 20-60 min 2-100 h

The train-on-the-track model

Rate = Number of tracks x Number of trains x Velocity of trains / Track length

GENE (DNA)

mRNA

Pro

tein

Pro

duct

RNA polymerase

Ribosome

Critical Factors:

For transcription – the attachment rate, since the number of gene copies (one or two), transcription velocity and length are fixed.

For translation – the number of mRNA's present, since the ribosomal attachement rate, translation velocity and length are fixed.

Summary of Steps in Eukaryotic Processing

What is the RNAP “train starter”? Transcription factors.

InducersRepressors

These are protein molecules, made by genes, that bind to a gene at an operator site, in or near a promoter region, upstream of where transcrip-tion takes place. They often exist in two forms quiescent and active. Usually a small molecule induces the change:

Inactive factor small molecule active factor

Transcription FactorsIt is important to remember that transcription factors are

proteins, come from genes (like all proteins), and may influence either their predecessor gene or –often– other genes.

Summary of the structure of the Engrailed homeodomain bound to DNA, as revealed by X-ray crystallography. Cylinders represent the three -helices of the homeodomain, ribbons represent the sugar phosphate backbone of the DNA and bars symbolize the base pairs. The recognition helix (3) is shown in red.

Transcription factors have many shapes

and thus modes of interaction

with DNA

Promoters (pol II)

Contain multiple binding sites for transcription factors.

Other binding sites upward, downward of (enhancers), and within transcribed region.

Basal Transcription Factors

TATA box – TATAa/tAa/ t Initiator – YYANa/ tYY,

where Y is a pyrimidine, N is any base, and transcription begins at the A

Picture shows assembly of basal pol II on adenovirus ML promoter

Preassembly of the Complex is possible: kinetic implications?

Actual Initiation (White, p. 62)

Promoter melting – DNA strands separate.Initiation – first RNA phosphodiester bond is

formed.Clearance – pol II released from the factors

assembled at the promoter.

Elongation and TerminationElongation factorsTermination Sequence (AAUAAA) and cleavage

factor. Polyadenylation.

Enhancers (White, p. 73)

Enhancer region located ~ 1 kb upstream of mouse muscle creatine kinase gene. There are at least 6 different transcription factors with expression “governed by combinatorial interactions amongst the transcription factors”.

Transcription factor activation

Transcription Factor Production, one example. (White, p. 170)

Transcription Control by Stimulated

Translocation

Transcription of the WT1 Gene

Negative feedback: WT1 protein inhibits expression of its own gene and also that of PAX-2 an activator of th WT1 promoter.

MyogenesisUpstream regulators force differentiation to mesodermal precursor cells that then express bHLH proteins that stimulate transcription of their own genes. They also activate genes that make MEF2, which further accelerates transcription of genes for bHLH proteins. MEF2 and bHLH proteins both stimulate other muscle-specific genes.

Positive feedback!

In general, transcription factors and the molecules that activate them are crucial to determining the array of genes that are on.

Transcription factors determine mRNA production;

do they determine mRNA number?

(mRNA number is what determines translation)

mRNA number is determined by a balance between generation and

consumption rate:

(mRNA)Production rate (mRNA)

dk

dt

This is the first part of the Goodwin equation set.

mRNA numbers determine protein production; do they

determine translation product levels?

Translation product levels are determined by a balance between their generation and consumption

rates:

(product)Production rate (product)

First-order decay processes are almost always

assumed. The decay constant, however, may be

fixed by other situations within the cell.

dk

dt

This is the second part of the Goodwin equation set.

Frequently a translation product will mediate the level

of another metabolite (galactosidase, tryp enzymes)

This can give rise to a third set of Goodwin equations.

Product levels determined by the third set often provide the "loop-closing" feedback.

What has been covered?

Transcription factor Active Gene

Transcription Product:

Messenger RNA

Translation Product:

(Active Protein)

Change in Change in

transcription factorcofactor level

activity