Transcriptional control

Coordinately regulated genes are not linked in eukaryotes In prokaryotes, several genes encoding for

particular proteins will link together in an operon.

When response to the activating signal, the gene will be transcribed as one single polycistronic mRNA. Translation of the mRNA will result into individual proteins following binding of the ribosome at particular sites. Eg: lac operon where three genes encoding for three different proteins are linked together and are induced in the presence of lactose

In eukaryotic organisms , this type of linkage does not occur. Individual genes are transcribed by RNA polymerase II into individual monocistronic mRNA encoding single protein. Proteins that needed to be together will not be linked and therefore expression need to be coordinated.

Genes encoding these proteins are not closely associated. Often present in different chromosome in that particular nucleus. Example the production of antibody by mammalian B cell. Need to synthesize light chain and heavy chain to form the antibody molecule/immunoglobulin.

Genes for antibody:

light chain in chromosome 2 and 22. heavy chain in chromosome 14.

Therefore, greater flexibility needed to regulate gene expression in eukaryotes due to this factor

The Britten and Davidson model (1969)

This is a model to explain gene expression at transcriptional level. They proposed that genes regulated in parallel with one another in response to a particular signal contain a common regulatory element. Gene will be activated in response to the signal.

Individual genes could contain more than one regulatory element. Some regulatory element would be shared with other genes. Specific signal causing gene activation where it will stimulate specific integrator gene. The product of the integrator would activate all the genes containing one particular sequence element.

This mechanism allow the regulation of gene at distinct region. In modern terms, the integrator gene would be considered as transcription factor which binds to regulatory sequences and activates expression of the corresponding genes. It is clear now that the target DNAbinding sites for transcription factors are short sequence approximately 10-30bp in length.

Short sequence element

The sequences that play role in regulation of eukaryotic transcription would be located upstream of the transcription start site within or adjacent to the promoter. For example in heat shock protein. Gene encoding this protein is called hsp70 with molecular weight of 70kDa

Exposure of a wide variety of cells to high temperature will result in the synthesize of heat shock protein , the most abundant is hsp70. Sequence present in the upstream region of hsp70 gene are also found in other genes. The sequences are found shared by :

The 70kDa heat-shock protein gene

hsp70 gene found in heat-inducible genes. Other non heat-inducible genes.

Name

Consensus

Other gene containing sequence

TATA box CCAAT box

TATAA/TAA/T In many genes TGTGGCTNNNAG and -globin, CCAA herpes simplex virus thymidine kinase GGGCGG Metallothionein II A. type II procollagen Somatostatin, fibronectin Collagenase, class I antigen H-

Sp1box

CRE AP2 box

T/G T/A CGTCA CCCCAGGC

The CCAAT and Spl boxes

The basal transcriptional complex containing TBP, RNA polymerase II and other associated factors produce low rate of transcription. Rate is enhanced by binding of other transcription factors upstream of the TATA box. The binding sites of these factors are present in wide variety of gene with different activity.

Example is the Spl box, two copies of which are present in the hsp70 gene promoter The GC rich DNA sequence binds a transcription factor called Spl which is present in all cell types. Another example is CCAAT box which is present upstream of the transcription site in many genes. These boxes is believed to be involved in regulation of transcription by binding to

The heat shock element

There are sequence which is shared only by the hsp70 gene. This sequence is found 62 bases upstream of the start site of transcription in most heat-inducible genes. This heat shock element (HSE) is believed to play a critical role in the transcribing of the hsp70 gene.

To prove this, Pelham (1982) transferred the HSE on to the nonheat inducible thymidine kinase (tk) gene taken from the eukaryotic virus, herpes simplex. When hybrid gene introduced into cells and temperature raised, increased thymidine kinase production was detected. HSE made the tk gene inducible by elevated temperature

Heat inducible Drosophila hsp 70 gene hsp 70 Heat shock Consensus element

Non inducible

HSV thymidine kinase gene tk

Chimaeric gene

tk

Introduced into cells and raise temperature

tk Heat inducible transcription

e 1:Heat-shock consensus element ates heat inducibility. Transfer of this sequence gene (thymidine kinase) which is non inducible ers this gene heat inducible

enhancer

Regulatory sequences that act at a distance. Is an enhancer is linked to a promoter,the activity of the promoter can be increased several hundred fold. The position and orientation of enhancer results to conclusion of its action:

Enhancer can activate promoter when placed up to several thousand bases from the promoter. Enhancer can activate promoter regardless of its orientation.

Distance enhancer a) distance

promoter Transcription unit

enhancer b) orientation enhancer

promoter Transcription unit promoter Transcription unit

enhancer c) position

promoter Transcription unit

Transcription unit promoter Transcription promoter

enhancer unit

enhancer

Tissue specific enhancer

A tissue-specific enhancer can activate the promoter of its own or another gene only in one particular tissue and not in others. Tissue specific enhancers have been detected in genes expressed specifically in liver (-fetoprotein, albumin, -1-antitrypsin) and also the endocrine and exocrine cells of the pancreas (insulin, elastase, amylase).

In the case of insulin gene, 250 bases upstream regions of the transcriptional start site being crucial in producing high level expression in pancreatic endocrine cells. This position corresponds to the tissue specific enhancer. Therefore this region is important in gene regulation. Mutation of the conserved region abolishes the production of genes that is usually expressed in that particular tissue.

The enhancer and promoter of insulin gene was linked to gene encoding the large T antigen of the eukaryotic virus SV40. Production can be measured using specific antibody. The hybrid was introduced into a fertilized mouse egg and egg was return to the oviduct of the mouse. Expression of large T antigen analysed in all tissue of the transgenic mouse. Expression of large T was detected in pancreas and NOT in other tissue Indication of tissue specific activity of the insulin gene enhancer

Enhancer being silencer

Some sequence act like enhancer but the other way round. It silent the transcription. Example in collagen type II, growth hormone, glutathione transferase. Like enhancers, silencers can act on distant promoters when present in either orientation but have an inhibitory rather than simulatory effect on the gene expression.

It acts at chromatin structure by recruiting factors which direct the tight packing of adjacent DNA or by binding protein which will then inhibit transcription. Or by binding negatively acting transcription factors. Whether acting positively or negatively, enhancers play a critical role in the regulation of eukaryotic gene expression.