DAIRY BIOTECHNOLOGY

Module 2. Fundamental biological principles

Lesson 6
GENE EXPRESSION – TRANSCRIPTION AND TRANSLATION

6.1 Introduction

Gene is defined as a specific nucleotide sequence encoding a particular function through synthesis of a protein. It represents the simplest hereditary unit in a cell which is associated with regulatory, transcriptional and other functional regions. In general these genes hold the information to build and maintain an organism's cells and pass genetic traits to offspring. All the metabolic functions in a cell are performed by expression of Genes. Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins which can serve as the structural component of the cell and biological catalysts to facilitate cellular metabolic reactions. rRNA or tRNA constitute the non-protein component of the coding genes. The process of gene expression is used by prokaryotes, eukaryotes and viruses to generate the macromolecular machinery for life. The flow of hereditary information from genes to proteins is dictated by ‘Central Dogma of Life’.

6.2 Central Dogma of Life

The central dogma of life was first visualized by Francis Crick during the year 1958 which refers to the fact that hereditary information normally travels in one direction only i.e. from DNA to DNA (replication) to RNA (transcription) and to Protein (translation) as shown in Fig. 6.1. However, the reverse flow of information from RNA to DNA is also possible and the process is called Reverse Genetics.

Fig. 6.1 Central dogma of life

The process of gene expression involves two major steps i.e. transcription and translation as defined below:

6.3 Transcription

Transcription is the process by which the genetic information from one strand of DNA is transferred to mRNA which is eventually used as template in the protein synthesis. This is the key step involved in gene expression.

One of the DNA strands which provides information for synthesis of mRNA is called template or anti-sense or non-coding strand. The other strand that matches exactly with the mRNA is called coding strand or sense strand or non-template strand .

The process of transcription involves the following steps:

6.3.1 Initiation

Initiation begins with the formation of transcription initiation complex with the binding of DNA dependent RNA polymerase (multi subunit complex) at promoter sequence. Promoter sequence is a specific base sequence on a DNA which serves as a binding site for RNA polymerase enzyme. The length of the sequence is typically 20-40 bases. The sequence helps in indicating the correct beginning of a gene which is being transcribed. In bacteria, this region contains two short regions –10 (TATAAT) and –35 (TGTACA). These sequences are recognized by sigma factor of RNA polymerase.

RNA polymerase enzyme unwinds a short section of the DNA double helical structure near the start of the gene and forms a transcription bubble. Once the DNA molecule unwinds, RNA polymerase begins the synthesis of the mRNA using the information of the template strand. Once ~ 9 – 10 bases long mRNA is synthesized, the process enters in the elongation phase.

6.3.2 Elongation

RNA polymerase moves along the DNA in 5′→3′ direction adding ribonucleotides to 3′-OH group of growing RNA chain until termination.

6.3.3 Termination

When RNA polymerase reaches a termination sequence, transcription stops either by rho dependent or rho independent process followed by the release of mRNA and RNA polymerase from the DNA molecule.

The newly synthesized mRNA is used as a template for protein synthesis.

6.4 Translation

Translation is the process of decoding the information contained in the form of codon sequences on mRNA and synthesizing a chain of amino acids which finally results into a protein molecule.
The process of translation involves the following steps:

6.4.1 Initiation

Initiation involves the binding of small subunit of ribosome (30S) to a specific sequence called Ribosomal Binding Site (RBS) or Shine-Dalgarno (SD) at the 5' end of mRNA. The larger subunit of ribosome (50S) binds with initiation complex. The structure of prokaryotic ribosome is shown below in Fig. 6.3.

Fig. 6.3 Sructure of prokaryotic ribosome

Now, a special acylated tRNA molecule carrying the amino acid methionine binds with pre-initiation complex forming the initiation complex. The cloverleaf structure of tRNA showing acceptor arm for amino acid and the anti codon loop for codon recognition is illustrated in Fig. 6.4.

The function of tRNA is to transfer the appropriate amino acids from the cytoplasm to the new polypeptide chain, which is being constructed, on the ribosome.

Proper positioning of the mRNA is vital to determine its reading frame, which defines which group of three bases is to be read as codons. For example, AUG UCC UGG or A UGU CCU GG. Hence, the presence of a start codon AUG coding amino acid methionine signals beginning of genetic message. The translation initiation process is shown in Fig. 6.5.

6.4.2 Elongation

After completion of the initiation step i.e. 70S initiation complex, ribosome proceeds to the elongation phase of protein synthesis. The methionine containing tRNA occupies directly the Peptidyl “P” site on the ribosome and a new tRNA carrying the next amino acid occupies the Acceptor “A” site. The Second amino acid forms a peptide bond with the previous amino acid with the help of peptidyl transferase. The tRNA carrying the dipeptide moves to the P site emptying the A site in the process, for accommodating the new tRNA molecule carrying the next amino acid. This process is repeated with sliding of mRNA along the ribosome three bases at a time and peptide chain growing with one amino acid at a time. The detailed elongation process has been shown in Fig. 6.6 A .

6.4.3 Termination

There are three termination codons that are employed at the end of a protein-coding sequence in mRNA: UAA, UAG, and UGA. There are no tRNAs that can recognize these codons. In the absence of such specific tRNA, one of several proteins, called release factors, binds and facilitates release of the mRNA from the ribosome and subsequent dissociation of the ribosome. Thus termination codons act as the final punctuation in the message of mRNA by telling cellular machinery that the product is complete and to stop adding amino acids. The process of termination & translation are shown in Fig. 6.6 B &.Fig. 6.7.

Further Reading

Books

Berg J, Tymoczko JL, Stryer L (2006). Biochemistry (6th ed.). San Francisco: W. H. Freeman. ISBN 0716787245

Fundamental Bacterial Genetics, Nancy Trun, Janine Trempy (Eds), Wiley-Blackwell, 2003, ISBN: 978-0-632-04448-1

Molecular Biology of the Gene, Sixth Edition, James D. Watson (Editor) Cold Spring Harbour Press and Benjamin Cummings, ISBN 978-080539592-1

Molecular Biotechnology - Second Edition, S. B. Primrose, Blackwell Science Inc., ASIN: 0632030534

DNA and Biotechnology, Fitzgerald-Hayes, M. And Reichsman, F. 2^nd Amsterdam : Elsevier, 2010. ISBN : 0-12-048930-5

Internet Resources

http://en.wikipedia.org/wiki/Transcription_(genetics)

http://en.wikipedia.org/wiki/Translation_(genetics)