SCBC203 Gene Expression. Assoc. Prof. Rutaiwan Tohtong Department of Biochemistry Faculty of Science PR318

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1 SCBC203 Gene Expression Assoc. Prof. Rutaiwan Tohtong Department of Biochemistry Faculty of Science PR318 1

2 Gene Expression Gene expression is a process where by the genetic information stored in the gene in the form of DNA, is expressed or read, such that proteins are made and the amino acid sequences specified by the instructions (base sequences) stored in the DNA. Gene expression is consisted of 2 processes, transcription and translation.

3 Objectives 1. Explain what gene expression is 2. Explain the biological roles, significance, key principles & components of transcription 3. Compare & contrast DNA and RNA synthesis 4. Compare & contrast transcription in Prokaryote vs Eukaryote 5. Explain the biological roles, significance, key principles & components of translation 6. Describe what Wobble Hypothesis is, & its significance 7. Compare & contrast translation in Prokaryote vs Eukaryote

4 Outline 1. RNA synthesis (Transcription): The principle 2. Transcription: Where? When? How? 3. The gene structure and the promoter region 4. Transcription initiation & termination 5. RNA processing 6. Comparison between Prokaryote vs Eukaryote 7. Inhibitors of transcription 8. Protein synthesis (Translation): The principle 9. Translation: Where? When? How? 10. Components of translation 11. Comparison between Prokaryote vs Eukaryote 12. Wobble Hypothesis 13. Inhibitors of translation

5 RNA synthesis (Transcription) The principle RNA synthesis using DNA as a template Occurs only on 1 of the 2 DNA strands at anytime 5 to 3 polymerization of the new chain Complimentary & anti-parallel to the template strand Use of U instead of T From transcription start site to transcription termination site RNA synthesis is the production of RNA using DNA as a template. It is a part of the gene expression process which is required for the maintenance of life. Hence, in contrast to DNA synthesis, RNA synthesis occurs almost constantly through out the life time of an organism, although the rate of which varies according to the developmental or physiological state of the cells.

6 Promoter Terminator Transcription: Where? When? How?

7 The Products of Transcription

8 The Products of Transcription Transcription in eukaryotic cells result in pre-mrna, which is consisted of exons & introns. The pre-mrna undergoes RNA processing to yield a mrna which is now ready to be transported out of the nucleus into the cytoplasm, where it will be translated into proteins.

9 Preparation for transcription occurs at the gene Promoter Promoter Transcription start site Transcription termination site The haploid genome of human contains 3 billion bp of DNA, only <2% of which is the coding sequences. The number of protein-coding genes are about 20,000.

10 The Promoter The promoter serves as the binding sites for RNA Polymerase & transcription factors when they prepare to start the transcription process. The promoter usually contains patches of conserved sequences which are recognized & bound by RNA Polymerase & transcription factors.

11 Three-Step- Transcription Initiation

12 Elongation RNA synethesis using DNA as a template Occurs only on 1 of the 2 DNA strands at anytime 5 to 3 polymerization of the new chain Complimentary & anti-parallel to the template strand Use of U instead of T From transcription start site to transcription termination site

13 Termination Rho-dependent Rho-independent Rho-dependent transcription termination: This mechanism requires the presence of rho protein, a RNA-binding protein that forms a hexameric complex at a particular signal site, and races up the RNA strand. Once it reaches the polymerase, the protein complex displaces the polymerase from the DNA, terminating transcription. Rho-independent transcription termination: This mechanism does not require rho protein (hence the name), and requires two elements; a hairpin formation (complementary sections of the transcribed RNA strand), followed immediately by a long stretch of T or A on the template. The hairpin formation causes RNA polymerase to slow or stall; normally, though, the polymerase can work through such an block (the diagram shows the hairpin as GC rich; this just makes it harder to work through, because GC makes three hydrogen bonds rather than two, and so the hairpin is more stable). If it is coupled with a long stretch of U or A, this destabilizes polymerase even further; not only is the T-A bond weaker, but the repeated usage of these nucleotides depletes the local concentration of them. Polymerase ends up waiting longer and longer for the next NTP, until it spontaneously dissociates.

14 DNA-dependent RNA Polymerases

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16 RNA Processing Only eukaryotic primary transcripts undergo 3-step RNA processing to produce mrna which will be transported from the nucleus to be translated into proteins in the cytoplasm

17 5 CAP 3 Poly-A tail

18 RNA Splicing The product of eukaryotic transcription is the Primary transcript (Pre-mRNA). They must undergo RNA Splicing or Cutting, one of the 3 steps in RNA processing, to remove introns and to join together exons. Each primary transcript may undertake > 1 way of splicing, leading to various combinations of introns spliced out, and exons joining together, resulting in >1 protein species produced from a single transcript.

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20 Rifampicin is a semisynthetic antibiotic produced from Streptomyces mediterranei. It has a broad antibacterial spectrum, including activity against several forms of Mycobacterium. In susceptible organisms it inhibits DNA-dependent RNA polymerase activity by forming a stable complex with the enzyme. It thus suppresses the initiation of RNA synthesis. Actinomycin D is derived from STREPTOMYCES parvullus. It binds to DNA and inhibits RNA synthesis (transcription), with chain elongation more sensitive than initiation, termination, or release.

21 Protein synthesis (Translation) The principle Protein synthesis is the production of polypeptides by joining amino acids via peptide bonds, using RNA as a template. It is the latter half of the gene expression process, occuring almost constantly through out the life time of an organism. Protein synethesis using mrna as a template mrna template is read from 5 to 3 Polypeptide chain growth from N to C termini The sequence of amino acids that are put together is determined by the sequence of base pairs of mrna 3 bp = 1 aa, Start & Stop codons

22 Protein synthesis (Translation) The principle mrna, trna, rrna, Ribosomes, Translation Factors, ATP/GTP Codon-anticodon Peptide bond formation Need free NH 2 group on the incoming aa-trna

23 P = Polypeptide A = Aminoacyl E = Exit Translation: Where? When? How?

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26 The Ribosomes Ribosomes are found floating freely or bound to ER. Those free ribosomes synthesize cytoplasmic proteins, where as ER-bound ribosomes synthesize proteins to be exported or membrane-proteins

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28 trna trna is charged with amino acids on its 3 end and carries it to the site of polymerization in the ribosome. It has the characteristics clover leaf shape with an anti-codon loop used for forming complimentary base-pairing with codons of the mrna.

29 trna trna is characterized by the presence of modified amino acids dispersed through out the molecule. These modified amino acids influence the efficiency of trna charging and codon-anti-codon pairing properties.

30 Translation begins at start codon (AUG) and ends at stop codon (UGA, UAG, UAA)

31 Prokaryote vs Eukaryote Translation

32 Mechanism of Translation Start in Prokaryote vs Eukaryote Start amino acid in Prokaryote vs Eukaryote Prokaryote Eukaryote Met is the start codon for Eukaryotes, where as f-met is the start codon for Prokaryotes. Prokaryotes localizes the start codon using SD sequence in the mrna to bind to 16s rrna of the small subunit ribosomes, where as Eukaryotes uses 40s ribosome to scan from 5 cap to the first start codon it encounters.

33 Wobble Hypothesis

34 Non-Watson/Crick base-pairing is permissible at the 3 rd base of codon and the 1 st base of anti-codon only

35 Inhibitors of Protein Synthesis

36 References ISBN-10: ISBN-13: