Gene Expresssion


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Gene Expresssion

  1. 1. MIC210 BASIC MOLECULAR BIOLOGY Lecture 3 Gene Expression By SITI NORAZURA JAMAL (MISS AZURA) 03 006/06 483 2132
  2. 2. Outline 1. Gene expression in prokaryotic cells – DNA to mRNA to protein. 2. Gene expression in eukaryotic cellsIntron splicing, 5’ capping, 3’-poly-A tail 3. DNA Replication 4. Reverse transcription
  3. 3. Every cell has the same DNA and therefore the same genes. But different genes need to be “on” and “off” in different types of cells. Therefore, gene expression must be regulated.
  4. 4. Gene expression must be regulated in several different dimensions— In time: 6 mos 14 wks 1 day 12 mos 10 wks 18 mos At different stages of the life cycle, different genes need to be on and off.
  5. 5. 1) Gene expression in prokaryotic cells– DNA to mRNA to protein.
  6. 6. 1. Gene expression : DNA to mRNA to protein • Gene expression – process where the information in a gene is read and used to synthesize a protein • Genetic information is linearly transferred from DNA to protein. • What proteins you can make depends on what genes you have Gene expression in prokaryotes
  7. 7. Transcription • a messenger RNA (mRNA) molecule is synthesize using the antisense strand as a template • the genetic information is now transferred to the mRNA • RNA is like DNA except : ribose sugar, single stranded, uracil
  8. 8. Molecular Components of Transcription • RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotides • RNA synthesis follows the same base-pairing rules as DNA, except uracil substitutes for thymine • The DNA sequence where RNA polymerase attaches is called the promoter; in bacteria, the sequence signaling the end of transcription is called the terminator • The stretch of DNA that is transcribed is called a transcription unit Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings
  9. 9. Translation • the information in the mRNA is read in a set of 3 bases – a codon • each codon codes for an amino acid • a chain of amino acids – a polypeptide – is built by reading the codons • all these happen in the ribosome in the cytoplasm
  10. 10. 2) Gene expression in eukaryotic cells- Intron splicing, 5’ capping, 3’-poly-A tail
  11. 11. The story is much more complicated in Eukaryotes
  12. 12. Important differences • A ‘cap’ is added to the 5’ end of the mRNA • A polyA tail is added to the 3’end • Introns are removed by a process called splicing
  13. 13. Introns and mRNA splicing • Most eukaryotic genes contain introns and exons • Exons are DNA sequences that carry genetic information • Introns do not carry genetic information • the introns are removed from the mRNA by a process called splicing • whereby the introns are cut out – and the exons are rejoined • this mature mRNA is then translated to make proteins
  14. 14. 3) DNA Replication
  15. 15. 3. DNA replication Every new cell must have a complete set of genes Before cell division occurs, the DNA is replicated so that each new cell has its own set of DNA
  16. 16. Overview Synthesis of the leading strand during DNA replication Origin of replication Leading strand Lagging strand Primer Lagging strand Leading strand Overall directions of replication Origin of replication 3 5 RNA primer 5 “Sliding clamp” 3 5 Parental DNA DNA poll III 3 5 5 3 5
  17. 17. In general : • the original DNA molecules to serve as a template • the new DNA strand is synthesized by the enzyme DNA polymerase III • Complementary base pairing ensures that the sequence of the template is copied accurately 3‟ T T T 5‟ DNA polymerase III New DNA strand
  18. 18. Synthesis of new DNA strand requires a primer and can proceed only in a 5’  3’ direction (why?) 5’ PO4 In the cell, the primer is a short RNA molecule In the test tube, a short piece of DNA will also work.
  19. 19. DNA replication step-by-step 1) Double helix structure opened up by a helicase enzyme The single stranded regions are stabilized by SSBs (single stranded binding proteins)
  20. 20. DNA replication step-by-step 2) Another enzyme, primase, makes a short RNA primer
  21. 21. DNA replication step-by-step 3) Then DNA polymerase III begins to extend the new DNA strand
  22. 22. DNA replication step-by-step Direction of replicasome 4) All these enzymes work together in a complex known as a replicasome. The replicasome moves in one direction, following the replication fork
  23. 23. The two strands of a DNA are not equal (when it comes to replication) Replication can only happen in a 5‟ to 3‟ direction „leading‟ and „lagging‟ strands
  24. 24. DNA replication step-by-step On the leading strand, everything’s OK - DNA synthesis occurs continuously in a 5’  3’ direction
  25. 25. DNA replication step-by-step On the lagging strand, we have a problem - DNA synthesis cannot happen in a 3’  5’ direction - thus, multiple primers are made - new DNA is synthesized as small Okazaki fragments (5’  3’) - the primers are then replaced with DNA by DNA polymerase I - the DNA fragments are then joined by DNA ligase
  26. 26. Fig. 16-17 A summary of bacterial DNA replication Overview Origin of replication Lagging strand Leading strand Leading strand Lagging strand Overall directions of replication Single-strand binding protein Helicase 5 3 Parental DNA Leading strand 3 DNA pol III Primer 5 Primase 3 DNA pol III Lagging strand 5 4 DNA pol I 3 5 3 2 DNA ligase 1 3 5
  27. 27. 3 Synthesis of the lagging strand 5 5 Template strand 3 3 RNA primer 3 1 5 5 3 1 5 3 5 2 3 3 5 Okazaki fragment 3 5 1 5 3 5 2 1 5 3 1 2 Overall direction of replication 3 5
  28. 28. Proof reading minimized replication error DNA polymerase III has a 3‟  5‟ exonuclease activity that can cut and repair mistakes Remember : DNA replication has to be very accurate (or else?)
  29. 29. DNA replication is semi conservative Replication : From one DNA molecules to two Identical sequences
  30. 30. 4) Reverse Transcription
  31. 31. 4. Reverse transcription – from RNA to DNA
  32. 32. The transfer of genetic information from RNA to DNA • By the enzyme reverse transcriptase found in retrovirus • This allows us to make cDNA (complementary DNA) from mRNA • and obtain a gene sequence without the introns Reverse transcription cDNA