Question 2- The first gene to be cloned and expressed in E. coli was the insulin

ID: 268245 • Letter: Q

Question

Question 2- The first gene to be cloned and expressed in E. coli was the insulin gene. This was no easy feat. What differences between prokaryotes and cukaryotes would the investigators have to overcome when expressing a human gene from a plasmid in a prokaryotic cell? Please include issues of transcription, processing, translation, and post- translational modifications. This question is meant to help you understand regulation of eukaryotic genes. Please make sure that you answer this thoroughly. It should be at least 2 bluebook pages. s)

Explanation / Answer

To clone eukaryotic gene in E. coli, or any suitable prokaryotic organism, and to express a eukaryotic gene in prokaryotic system, it is essential to incorporate the indispensable factors associated with eukaryotic, transcription of DNA to mRNA, translation to express protein and post translational modification of proteins.

The different features of eukaryotic systems essentially required for transcription, translation, post-translational modifications, and their difference from prokaryotic system are:

E. coli:

Lacks, nucleus, transcription translation occur simultaneously i cytoplasm

Eukaryotes:

Has nucleus: Transcription occurs in nucleus followed by translatio and modifications in cytoplasm.

1. RNA Polymerase-

Single type of RNA used for transcription of all gene.

Three separate RNA polymerases:

a. RNA pol I- synthesizes rRNA.

b. RNA pol II- synthesizes mRNA.

c. RNA pol II- synthesizes tRNA and other small RNAs.

2. Gene structures- Cistrons

Usually polycistronic. Multiple gene products.

Operon systems or model controlling each transcriptional unit separately.

Usually monocistronic. Single gene product from mRNA, which are later cleaved ad modified.

3. RNA processing

RNA processing is typical feature of eukaryotes, includes:

a. Capping- modification of pre-mRNA at 5’ end, by methylation of Guanine at 7’ position.

B Poly A tail- Ploy Adenylation (A-A-A-A) at 3’ end of mRNA.

4. Split gene structure and splicing-

Most prokaryotic sequences (of gene, RNA, proteins) are contiguous.

Many eukaryotic gene contain, regions of “expressed sequence” or exons, which code for proteins and non-coding or” intervening sequence” or introns.

The pre-RNA transcript requires slicing process, by which introns are removed, exons are joined to form mature RNA.

The process of splicing is mediated by small nuclear ribonuclear proteins (snRNPs or SNURPS)

Some eukaryotic genes may undergo alternative splicing, like fibronectin.

5. Promoter

Short sequences- -10 and -35 upstream of transcription start site.

The -10 region, usually contains of TATAAT, referred to as Pribnow box

Often are tripartite, consisting of- GC box, CAT, TATA box.

Initiation requires binding of transcriptional factors, like TFIIA, TFIIB, etc, binding to TATA box.

6. Regulatory factors

Controlled by operon system, with proteins inducers/ repressors

Enhancers- There are several short sequences, that allows binding of transcription factors, that in turn regulate the transcription. These include: a) enhancers- “turn on” transcription (binds activators), b) silencers- - “turn off” transcription (binds repressors).

Enhancers allow the binding of transcription factors, referred to as enhancer binding proteins. The enhancer may be situated, upstream downstream or within the gene to be expressed. Enhancers operate as “cis acting regulatory elements”, that can promote the activity of the promoter, in a position independent manner. This is proved by observing that, enhancers may be functional at variable distance, closer distance (of 200 bp) or further away (even 25kb).

7. Translational factors

a.3 translational initiation factors, IF-I, IF-II, IFIII

b. Exit of deacylated tRNA fron E or exit site

a.10 identified translational initiation factors: eIF-I, eIF2, eIF3, eIF4, eIF4B, eIF-4C, eIF-4D, eIF-4F, eIF5, eIF6.

b. Exit of deacylated tRNA fron P site, as E site absent in eukaryotic Ribosome.

8. Posttranslational factors

Ia not common

Occurs, to target protein to varios part of the cells. Includes phosphorylation, glycosylation.