Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Fundamentals of Molecular Biotechnology
DNA, RNA, and Protein Synthesis
Conclusion

The DNA molecule consists of two antiparallel polynucleotide chains that form a double helix. Their monomeric unit is a nucleotide, which is composed of a nitrogenous base, deoxyribose, and a phosphate group. Adjacent NUCLEOTIDES in the chain are linked by phosphodiester bonds, while the chains are held together by Hydrogen Bonds formed between complementary bases. Specifically, adenine forms hydrogen bonds only with thymine, and guanine only with cytosine. The process of DNA duplication is called Replication, which involves a variety of Proteins, most notably DNA polymerases. Each DNA strand serves as a template for the synthesis of a complementary strand. Base complementarity between opposite strands ensures that the newly synthesized DNA is identical to the original one.

Proteins play a key role in carrying out all biological Functions. A protein molecule is a polypeptide composed of Amino Acids linked together by peptide bonds. The Amino Acid Sequence in a protein is determined by The nucleotide sequence of the DNA. RNA molecules (mRNA, rRNA, and tRNA), various Enzymes, and protein factors participate in Protein Synthesis. All RNAs are synthesized using DNA AS A template; this process is called METABOLISM/31.html">Transcription. The accuracy of transcription—its precise initiation and termination at the correct sites—is ensured by specific nucleotide sequences in the DNA and protein factors. In eukaryotes, most structural genes consist of coding regions (exons) and non-coding regions (introns). Primary transcripts contain both. However, upon completion of transcription, introns are spliced out and exons are joined together to form functional mRNA. The mRNA carries encoded information about The amino acid sequence of the corresponding protein molecule.

Protein synthesis is called Translation. tRNA and rRNA molecules play a crucial role in this process. A Cell contains more than 50 different tRNAs, each of which specifically binds one of the 20 amino acids via its 3' end. At the 5' end of the tRNA, There is a three-nucleotide sequence (the anticodon) that ensures the binding of the tRNA to the complementary three-nucleotide site on the mRNA molecule. There are two MAIN TYPES OF rRNA: small and large. They associate with the small and large ribosomal subunits, respectively—specialized structures where protein synthesis takes place. In prokaryotes, rRNA molecules are smaller than in eukaryotes.

In prokaryotes, translation begins with the binding of mRNA to the small ribosomal subunit. This is followed by the complementary base pairing of the first mRNA codon with the anticodon of the initiator tRNA (fMet-tRNA). The large ribosomal subunit then joins the complex, forming the initiation complex ready for Polypeptide chain synthesis.

In eukaryotes, translation begins with the attachment of the initiator tRNA, which carries a Methionine residue (Met-tRNA^Met), to the small ribosomal subunit; the mRNA also binds to this subunit via its 5' end. The small subunit migrates along the mRNA until it reaches the first AUG codon. This codon forms a complementary base pair with the UAC anticodon of the initiator tRNA. Next, the large subunit joins this complex, forming a ribosome ready for protein synthesis.

Elongation and termination of translation in pro- and eukaryotes are largely similar. Following The formation of the initiation complex, the next mRNA codon pairs with the anticodon of a tRNA carrying the corresponding amino acid (let us designate it as AA2). The first amino acid in the polypeptide chain, methionine, is cleaved from its tRNA and linked to AA2 via a peptide bond. The free tRNA^Met leaves the ribosome. The ribosomal complex moves along the mRNA molecule, and the peptidyl-tRNA—that is, the Met-AA2-tRNA^AA2 complex—occupies the site vacated by the departed tRNA. The next mRNA codon pairs with the corresponding anticodon of a tRNA carrying the amino acid AA3. AA2 is cleaved from its tRNA and linked to AA3 via a peptide bond, forming the Met-AA2-AA3-tRNA^AA3 complex. The amino acid-free tRNA leaves the ribosome. The ribosomal complex again moves along the mRNA molecule, and Met-AA2-AA3-tRNA^AA3 occupies the vacant site previously held by the preceding peptidyl-tRNA. These events repeat until the ribosome reaches a stop codon. None of the tRNAs possess an anticodon complementary to a stop codon. However, the stop codon is recognized by a release factor; upon binding of this factor to the ribosome, the bond between the final tRNA and the synthesized polypeptide is hydrolyzed, the tRNA, mRNA, and polypeptide are released, and the ribosome dissociates into its subunits.

The synthesis of mRNA and, consequently, protein synthesis must be strictly regulated because a cell lacks the resources for simultaneous transcription and translation of all structural genes. Both PROKARYOTES AND EUKARYOTES constantly synthesize only those mRNAs required for essential cellular functions. Expression of the remaining structural genes is under the tight control of regulatory systems that trigger transcription only when there is a demand for a specific protein or proteins. In prokaryotes, Transcription is initiated by the binding of RNA polymerase to the TATA and TTGAC sequences in the promoter region of a structural Gene or Operon. The turning on or off of certain operons is mediated by an effector that alters the conformation of the repressor protein, preventing it from blocking transcription. When the effector concentration in The Cell decreases, the repressor binds to a DNA region adjacent to the Transcription initiation site and obstructs the movement of RNA polymerase along the DNA molecule, thereby blocking transcription. In other operons, an activator protein binds to the DNA segment adjacent to the transcription initiation site, increasing The rate of transcription. The binding of an effector to the activator can decrease the rate of transcription. The DNA-Structure/156.html">Protein Interactions responsible for Transcriptional Regulation are highly specific to particular structural genes or operons. In eukaryotes, RNA polymerase II, which transcribes structural genes, associates with a whole set of proteins—transcription factors—that bind sequentially to the TATA sequence of the promoter region. Additional transcription factors, which bind to specific DNA sequences, are responsible for turning transcription on and off.

References

Buratowski S. 1994. The basics of basal transcription by RNA polymerase II. Cell 77: 1—3.

Kozak M. 1991. Structural features in eukaryotic mRNAs that modulate the initiation of translation. J. Biol. Chem. 266: 19867-19870.

Lodish H., D. Baltimore, A. Berk, S.L. Zipursky, P. Matsudaira, J. Darnell. 1995. Molecular Cell Biology. 3rd ed. Scientific American Books, Inc., New York, N.Y.

Nakamura Y., K. Ito, L.A. Isaksson. 1996. Emerging understanding of Translation termination. Cell 87: 147-150.

Schoenherr C.J., D.J. Anderson. 1995. The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron-specific genes. Science 267: 1360—1363.

Tate W.P., C.M. Brown. 1992. Translational termination: «stop» for protein synthesis or «pause» for Introduction/30.html">Regulation of Gene Expression. Biochemistry 31: 2443-2450.

Tjian R., T. Maniatis. 1994. Transcriptional activation: a complex puzzle with few easy pieces. Cell 77: 5-8.

Review Questions

1. Describe the overall process of DNA replication.

2. What are the differences between DNA and RNA?

3. Describe the Similarities and differences between prokaryotic and eukaryotic structural genes.

4. Describe the process of Polypeptide chain elongation.

5. What is the most probable nucleotide sequence encoding the following amino acid sequence: MAGGTWYQLFPRKMWNDSTLHPFILPMNVAG.

6. Which amino acid sequence corresponds to the following nucleotide sequence:

GCGAUCGACGAUGUUUCUAAAAGUAUCUСAUCGAAAUGAGGGUUCGUAAUAGCGACCCGGGCGG.

7. WHAT IS A TATA box?

8. What is an operon? What is its biological role?

9. Describe three different mechanisms of transcription regulation in prokaryotes.

10. Describe the key DNA elements responsible for the transcription of eukaryotic structural genes.