The Blueprint of Life, From DNA to Protein

1. The Blueprint of Life,From DNA to Protein Chapter 7

2. The Blueprint of Life • Characteristics of each cell dictated by information contained on DNA • DNA holds master blueprint • All cell structures and processes directed by DNA

3. Overview • Complete set of genetic information referred to as genome • Genome of all cells is composed of DNA • Some viruses have RNA genome • Functional unit of genome is the gene • Gene codes for gene product • Gene product is most commonly protein • Study of transfer of genes is genetics • Study of sequence of DNA is genomic

4. Overview • Living cells must accomplish two general tasks to multiply • DNA replication • DNA expression (gene expression) • Expression involves two process • Transcription • Copies information in DNA to RNA • Translation • Interpret RNA to synthesize protein • Flow of information from DNA to RNA to protein • Central dogma of molecular biology

5. Overview • Characteristics of DNA • Made up of deoxy-ribonucleotides • Nucleotides include: • Phosphate group • 5 carbon sugar • Deoxyribose • Nucleotides bond covalently between the 5’PO4 of one nucleotide and the 3’OH of another • Joining of nucleotides creates an alternating sugar-phosphate backbone

6. Overview • Characteristics of DNA • Each sugar (deoxyribose) molecule is connected to a nitrogenous base • Nitrogenous bases • Adenine (A) - purine • Thymine (T) - pyrimidine • Guanine (G) - purine • Cytosine (C) – pyrimidine

7. Overview • Characteristics of DNA • Chemical structure and joining of nucleotide subunits causes strands to differ at the ends • One strand has a phosphate attached at the number 5 carbon of the sugar. • Termed the five prime (5’) end • The other strand has a hydroxyl group attached to the number 3 carbon of the sugar. • Termed the three prime (3’) end

8. Overview • Characteristics of DNA • DNA occurs as double-stranded molecule • Strands are complementary to each other • Due to the specific base pairing of bases • A:T • C:G • Strands are held together with hydrogen bonds • Specific hydrogen bonding between bases • A is bound to T by two hydrogen bonds • G is bound to C by three hydrogen bond

9. Overview • Characteristics of DNA • DNA molecule is antiparallel • Strands are oriented in opposite directions • Strands differ at the ends • One strand oriented in the 5’ to 3’ direction. • The other strand is oriented in the 3’ to 5’ direction.

10. Overview • Characteristics of RNA • RNA is made up of nucleotides • Ribonucleotides • RNA contains nitrogenous bases • Adenine • Guanine • Cytosine • Uracil • Uracil replaces thymine in RNA • RNA usually exists as single stranded molecule

11. Overview • Characteristics of RNA • Portion of DNA acts of template for RNA synthesis • RNA molecule called transcript • Numerous transcripts can be produced from one chromosome • Either strand of DNA can act as template • Three different functional groups of RNA • Messenger RNA (mRNA) • Ribosomal RNA (rRNA) • Transfer RNA (tRNA)

12. Overview • Regulating the expression of genes • Nucleotide sequence codes for regulation mechanism for gene expression • Mechanisms determine duration of synthesis of gene products • Products are only made when required • Key mechanism is regulation of mRNA synthesis from DNA • Regulation of transcription

13. Bi-directional replication DNA Replication • DNA is replicated to create second copy of molecule • Molecule is identical to original • Replication is bidirectional • Replication begins at specific starting point • Proceeds in opposite directions • Allows replication to proceed more quickly

14. DNA Replication • DNA replication • The two strands are unwound and separated • Free, unbound nucleotides match up to the newly separated nitrogenous bases of the parent strand • The parent strand is also called the template strand

15. DNA Replication • DNA replication • Base pairing is specific in DNA replication • Where adenine is present only thymine binds in the new strand and vice versa • Where guanine is present only cytosine binds in the new strand and vice versa • Bases that are improperly inserted are removed and replaced with the correct base • Newly added bases are added by the enzyme DNA polymerase

16. DNA Replication • Specifics of DNA replication • As the strands of DNA unwind, it creates an area of replication called the replication fork • As nucleotides are added, the replication fork moves down the parental strand

17. DNA Replication • Specifics of DNA replication • DNA polymerase adds new nucleotides as they become available. • DNA polymerase can only add nucleotides to the free hydroxyl at the 3’ end • DNA polymerase replicates in 5’ to 3’ direction • Enzymes READS DNA template in 3’ to 5’ direction • Because of the antiparallel nature of the strands of DNA, the two new strands will grow in opposite directions • One strand is the leading strand • One strand is the lagging strand

18. DNA Replication • Specifics of DNA replication • Leading strand • Is synthesized CONTINUOUSLY as the DNA polymerase moves towards the replication fork • Lagging strand • Is synthesized DISCONTINUOUSLY in pieces as DNA polymerase moves away from the replication fork

19. DNA Replication • Specifics of DNA replication • DNA polymerase must bind to an RNA primer to begin synthesis • A second DNA polymerase removes any RNA primers • An RNA primer is required at each newly synthesized section of the lagging strand • DNA ligase joins the fragments of the lagging strand

20. DNA Replication DNA Replication • Specifics of DNA replication • Replication is completed when the replication fork reaches the end of the parent strands • The original parent strand and the newly synthesized daughter strand rewind • Each new strand of DNA consists of one parent strand and one daughter strand • DNA replication is referred to as semiconservative

21. Gene Expression • Involves two separate but interrelated process • Transcription • Process of synthesizing RNA from DNA template • Translation • RNA is deciphered to synthesize protein

22. Gene Expression • Transcription • Transcription is the synthesis of a strand of mRNA from a DNA template • mRNA carries the coded information from DNA to the ribosome, which is the site of protein synthesis • mRNA also plays an important role in translation

23. Gene Expression • Transcription • During transcription the enzyme, RNA polymerase, synthesizes a complementary strand of mRNA from a portion of unwound DNA

24. Gene Expression • Specifics of Transcription • RNA polymerase binds to a region of the DNA called the promoter • Only one strand of DNA acts as a template • This is called the sense strand • The strand not transcribed is the nonsense strand

25. Genet Expression • Specifics of transcription • Nucleotides in RNA are the same as those in DNA with one exception • Thymine is replaced with uracil • Binding in RNA is • A:U or U:A • C:G or G:C

26. Transcription Gene Expression • Specifics of transcription • RNA polymerase continues down strand of DNA until it reaches a site on DNA called the terminator • At the terminator RNA polymerase and the new strand of mRNA are released from strand of DNA

27. Gene Expression • Translation • Translation is the decoding of information held in the mRNA to synthesize proteins • Two more RNA molecules become involved in translation • Ribosomal RNA (rRNA) • Transfer RNA (tRNA)

28. Gene Expression • rRNA forms part of the ribosomal machinery used in protein synthesis • rRNA builds the ribosomes • tRNA recognizes specific sequences of mRNA and transports the required amino acids to form a polypeptide chain

29. Gene Expression • Translation • The language of mRNA is in the form of codons • Codons are groups of three nucleotides situated next to each other on DNA • Codons are written in terms of their base sequence in mRNA • The sequence of codons determines the sequence of amino acids in the protein

30. Gene Expression • Translation • There are 64 codons that make up the “alphabet” of proteins • Of the 64 codons, 61 are sense codons • Each coding a specific amino acid • The remaining 3 are nonsense codons • These code for termination of the message • Codons contained in mRNA are read into proteins through translation • The site of translation is the ribosome

31. Gene Expression • In response to each codon, tRNA brings the appropriate amino acid to the site of translation • Each codon has an anticodon • The anticodon is complementary sequence to the codon

32. Gene Expression • Translation • Ribosomes • The 30s and the 50s ribosomal subunits join together around the mRNA • The ribosomes direct the binding of tRNA to the correct codon on the mRNA • tRNA binds to the P site and the A site of the 50s ribosomal subunit • The ribosomes bind to the mRNA to be translated

33. Gene Expression • Specifics of Translation • The first tRNA binds to a start codon in the P site of the ribosome • AUG is the start codon for EVERY protein • AUG codes for the amino acid methionine • When the second tRNA binds to the A site, the amino acid of the first tRNA forms a peptide bond with the amino acid of the second tRNA P site A site

34. Gene Expression • Specifics of translation • After the peptide bond is formed between the two amino acids, the tRNA P site leaves the ribosome • The ribosome moves distance of one codon • Amino acid in the A site moves to the P site • A new tRNA fills the now empty A site

35. Gene Expression • Specifics of translation • The ribosome continues down the strand of mRNA • Amino acids form peptide bonds along the way • Translation is terminated when the ribosomes come to a stop or nonsense codon • At this point the ribosomes separate • The new polypeptide chain is released • The ribosome and the mRNA are free to begin translation again

36. Translation Gene Expression • Specifics of translation • As the ribosome moves down the strand of mRNA, the start codon is exposed • Once exposed, a new ribosome will attach and begin another polypeptide chain

37. Regulation of Gene Expression • Microorganisms posses mechanism to synthesize maximum amount of cell material from limited energy • Controls directed at metabolic pathways • Two general mechanism • Allosteric inhibition of enzymes • Controlling synthesis of enzymes • Directed at making only what is required

38. Regulation of Gene Expression • Principles of regulation • Not all genes subjected to regulation • Enzymes can be classified according to characteristics of regulation • Constitutive enzymes • Constantly synthesized • Enzymes of glycolysis • Inducible enzymes • Not regularly produced • turned on in certain conditions • Β-galactosidase • Repressible enzymes • Routinely synthesized • Generally involved in biosynthesis

39. Regulation of Gene Expression • Mechanisms controlling transcription • Often controlled by regulatory region near promoter • Protein binds to region and acts as “on/off” switch • Binding protein can act as repressor or activator • Repressor blocks transcription • Activator facilitates transcription • Set of genes controlled by protein is called an operon

40. Regulation of Gene Expression • Repressors • Control mechanism that inhibits gene expression and decreases the synthesis of enzymes • Repression is usually in response to the overabundance of an end product • Repression decreases the rate synthesis of enzymes leading to the formation of the particular end product • Regulatory proteins called repressors mediate repression • Repressors block the ability of RNA polymerase to bind and initiate protein synthesis

41. Regulation of Gene Expression • Activators • Control mechanism that turns on the transcription of a gene or set of genes • Inducers are substances that act to induce transcription • Enzymes synthesized in the presence of inducers are called inducible enzymes

42. Regulation of Gene Expression • Operon model of gene expression • An operon is a set of genes that includes an operator, promoter and structural genes • An operon is divided into two regions, the control region and the structural region • The control region include the operator and the promoter • This region controls transcription • The operator acts as the “on-off” switch • The structural region includes the structural genes • This region contains the genes being transcribed

43. Operator Gene 1 Gene 2 Gene 3 Promoter Operon structure Operator – binding site for the repressor protein for the regulation of gene expression Promoter – Binding site for RNA polymerase Structural Genes – DNA sequence for specific proteins

44. Regulation of Gene Expression • Lac operon • Example of induction of gene expression • Near the operon on the DNA is a regulatory gene called the “I” gene • This codes for the repressor protein • When lactose is absent, the repressor protein binds to the operator gene • Binding of the repressor gene prevents RNA polymerase from transcribing the structural genes • No mRNA is made and no enzymes are synthesized

45. Regulation of Gene Expression • Lac operon • When lactose is present the repressor binds to lactose instead of the operator • With the repressor bound to lactose, RNA polymerase is able to bind to the promoter and transcribes the structural genes • Lactose acts as an inducer by keeping the repressor from binding to the operator • It induces the transcription of the structural genes

46. Operator Gene 1 Gene 2 Gene 3 Promoter RNA polymerase Repressor Lac Operon Lac Operon 1. 2. 3. Lactose

47. Gene Expression and Environmental Fluctuations • Many organisms adapt to changing environments by altering level of gene expression • Mechanisms include • Signal transduction • Natural selection

48. Gene Expression and Environmental Fluctuations • Signal transduction • Process that transmits information from external environment to inside cell • Allows cell to respond to changes • Two-component regulatory systems • Relies on sensor and response regulator proteins • Sensors recognize change in environment • Response regulators activate or repress gene expression • Quorum sensing • Organisms sense density of population • Enables activation of genes beneficial to the mass

49. Gene Expression and Environmental Fluctuations • Natural selection • Mechanisms to enhance survivability • Antigenic variation • Alteration in characteristics of certain surface proteins • Example: Neisseria gonorrhoeae hides from host immunity by changing numerous surface proteins • Phase variation • Routine switching on and off of certain genes • Altering expression allows portions of population to survive and multiply