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10/21/13 Genetics: Chapter 7 What is genetics? • The science of heredity; includes the study of genes, how they carry information, how they are replicated, how they are expressed 1 10/21/13 Prokaryotic DNA replication, trascription and translation Gene Expression DNA DNA Replication Duplicates the DNA moleculeso its encoded information can be passed on to the next generation. Transcription Copies the information in DNA into RNA. RNA Translation Interprets the information carried by RNA to synthesize the encoded protein. Protein Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA structure: single subunit is a deoxyribonucleic acid—3 parts Adenosine NH2 N O O O O – P O P O P O – O – High-energy bonds O – N O CH2 N O N Adenine OH OH Ribose Where else have you seen this molecule? 2 10/21/13 RNA vs.DNACH OH O OH C O C H H C C OH H C C H C OH OH OH Ribose C OH C OH H H CH OH O O H C O C H H C C H H C C H C OH OH H Deoxyribose C OH C OH H H 5 2 1 H H H H 1 4 2 3 2 3 4 5 5 2 1 H H H H 4 2 1 3 3 4 5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Purines (double ring) Pyrimidines (single ring) N N H CH3 N H N H Adenine (A) (both DNA and RNA) O N N H N H N O O NH2 H N O H Thymine (T) (DNA only) H H N N H O H Uracil (U) (RNA only) NH2 H H NH2 H Guanine (G) (both DNA and RNA) N H N N O H Cytosine (C) (both DNA and RNA) 3 10/21/13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Sugarphosphate backbone Nucleobases G P 5' phosphate 1 4 3 2 A P 5 3 P 1 4 Nucleotide 5 2 C 5 1 4 3 2 T P 5 The 3' carbon of one nucleotide is linked to the 5' carbon of the next nucleotide via a phosphate group. 1 4 3 OH 2 3' hydroxyl Double stranded DNA forms a double helix 4 10/21/13 DNA double strand 5′ phosphate 5′ end 3′ hydroxyl 3′ end HO T C G A G Sugar C Nucleotide O O Sugar O P P T Sugar O Sugar P O P C Sugar Sugar O P DNA O P G Sugar O Sugar P O Sugar P A Sugar P Base pairs O P Notice the strands run in the opposite direction relative to eachother- Hydrogen bonds HO Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3′ hydroxyl 3′ end 5′ end 5′ phosphate 5 10/21/13 DNA is synthesized in one direction. New units added to the 3’ end only. So, direction is from the 5’ to 3’ BECAUSE, hydroxyl group is on the 3’ carbon of the pentose ring. DNA Replication 6 10/21/13 Enzymes necessary for DNA replication • • • • • Primase DNA Polymerase DNA gyrase DNA ligase Helicase DNA Polymerase adds nucleotides to the 3’ end Template strand 5′ DNA Replication 3′ T A C G G T A C T A G T A G T A G T C G A T T C G A A T C A T C A T C A G C T A A G C T T Direction of synthesis 3′ A 5′ DNA polymerase P P O A P O P P A G T C O O OH P O T O OH P New strand P Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 7 10/21/13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Replication forks 3′ 5′ 1 A helicase unzips the two strands of DNA. Helicase Leading strand 5′ 3′ 5 RNA primer DNA polymerase adds nucleotides onto the 3′ end of the strand. 5′ 3′ 5′ 2 Synthesis of the leading strand proceeds continuously as fresh template is exposed. 5′ 5′ 3′ Okazaki fragment of the lagging strand Primase synthesizes the RNA primer. 3 Synthesis of the lagging strand must be reinitiated as more template is exposed. Each time synthesis is reinitiated, a new RNA primer must be made. Discontinuous synthesis generates Okazaki fragments. 5′ 3′ 5′ 4 As DNA polymerase adds nucleotides to the 3′ end of one Okazaki fragment, it encounters the 5′ end of another. A different type of DNA polymerase then removes the RNA primer nucleotides and simultaneously replaces them with deoxynucleotides. 3′ 5′ 5 DNA ligase seals the gaps between Okazaki fragments by forming a covalent bond between them. DNA ligase 3′ 5′ 8 10/21/13 DNA replication: detail DNA replication…closer look 9 10/21/13 Gene Expression…why is it important? • Transcription • Translation Transcription: DNA to RNA • Requires an enzyme….. • RNA nucleotides • Base pairing rules for building RNA from a DNA template • Process proceeds in the direction 5 --->3 • Process begins at the promoter region and ends at the terminator sequence 10 10/21/13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Gene Expression Transcription Translation Transcription: RNA is transcribed from DNA 3′ Plus (+) 5′ strand GCTGATGAT CCGCGTAGGTGC T of DNA CGAC T AC T AGGCGC AT C C ACGA 5′ Minus (–) 3′ strand of DNA 3′ RNA 5′ GCUGAUGAUCC GCGUAGGUGCU Transcription: Promoter orients direction of transcription 11 10/21/13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Gene Expression Transcription Transcription: RNA synthesis Translation 5′ 3′ 3′ 5′ Promoter Terminator RNA polymerase 5′ 3′ 3′ 5′ 1 Template strand Sigma 5′ 2 3′ 3′ 5′ Promoter Initiation RNA polymerase binds to the promoter and melts a short stretch of DNA. 5′ RNA Elongation Sigma factor dissociates from RNA polymerase, leaving the core enzyme to complete transcription. RNA is synthesized in the 5′ to 3′ direction as the enzyme adds nucleotides to the 3′ end of the growing chain. A CUG G C T G AC 3 5′ 3′ 3′ 5′ Promoter Termination When RNA polymerase encounters a terminator, it falls off the template and releases the newly synthesized RNA. 5′ RNA polymerase dissociates from template. What are the possible products from transcription? • Messenger RNA (mRNA) • Transfer RNA (tRNA) • Ribosomal RNA (rRNA) 12 10/21/13 Fig. 7.3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Protein-encoding gene DNA rRNA gene tRNA gene Transcription Messenger RNA (mRNA) Ribosomal RNA (rRNA) Transfer RNA (tRNA) Translation Protein Quick check….. • Do we have a protein yet? • What have we made? • What is next? 13 10/21/13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Gene Expression Translation:RNA to Protein Transcription Translation Region translated 5′ 3′ mRNA Ribosome- Start binding site codon Stop codon Translation Protein Phe Ser His Cys Tyr Ser Pro Ala Gln Ser Met Tyr Gly Glu Val Leu Amino acid P ro • What is needed for the process? – mRNA – Ribosomes – Amino acids – tRNA Hydrogen bond tRNA Anticodon G G C C C G 5′ mRNA 3′ (a) Codon P ro tRNA G G C Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. (b) Anticodon 14 10/21/13 Translation: reading frame determines the protein Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Non-polar Amino acids H3 C CH3 H3 C CH3 CH2 CH3 C H H3 N+ C COO – H3 N+ C COO– H3 N+ C COO– H H Glycine (Gly; G) Alanine (Ala; A) CH3 CH H H3 N+ H Valine (Val; V) C COO– CH2 C H H2 C CH2 H3 N+ C COO– H2 N+ C COO– H H H Leucine (Leu; L) Isoleucine (IIe; I) Proline (Pro; P) CH3 H N S SH CH2 H 3N + CH2 C COO– H 3N + H Phenylalanine (Phe; F) CH2 H3C CH2 CH2 C COO– H 3N + CH2 C COO– H 3N + C COO – H H H Tryptophan (Trp; W) Cysteine (Cys; C) Methionine (Met; M) Polar/hydrophilic (uncharged) O O OH CH2 H 3N + C COO– C HO CH3 CH H 3N + H H 3N + OH NH2 C CH2 CH2 CH2 C COO– H Serine (Ser; S) N H 2 C COO– H 3N + H C COO– CH2 H 3N + H Threonine (Thr; T) Asparagine (Asn; N) Glutamine (Gln; Q) C COO– H Tyrosine (Tyr; Y) Polar/hydrophilic (charged) Basic NH2 C NH2+ Acidic NH3+ O – O O – O C CH2 H 3N + C COO– H Aspartic acid (Asp; D) HN CH2 C CH2 CH2 H 3N + C COO – H Glutamic acid (Glu; E) H 3N + C COO– H Histidine (His; H) CH2 NH CH2 CH2 CH2 CH2 CH2 CH2 H 3N + C COO – H Lysine (Lys; K) H 3N + C COO – H Arginine (Arg; R) 15 10/21/13 Fig. 2.13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. R Side chain— R is the general designation for a side chain H Amino group— positively charged at neutral pH O H N+ C C H H O– Carboxyl group— negatively charged at neutral pH The Genetic code 16 10/21/13 Structure of Ribosomes Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. f-Met P-site E-site Translation Initiation The initiating tRNA, carrying the amino acid f-Met, base-pairs with the start codon and occupies the P-site. A-site U A C A U G C C G U A C G A A G A U U A C U A G G A U 3′ 5′ mRNA f-Met Pro A tRNA that recognizes the next codon then fills the unoccupied A-site. U A C G G C A U G C C G U A C G A A G A U U A C U A G G A U 3′ 5′ Peptide bond f-Met Pro The ribosome catalyzes the joining of the amino acid carried by the tRNA in the P-site to the one carried by the tRNA in the A-site. 5′ U A C G G C A U G C C G U A C G A A G A U U A C U A G G A U 3′ (a) 17 10/21/13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. R R H H H O N+ C H H + C H O– Amino acid N+ C H H C O– Amino acid Dehydration synthesis H2O R R O H H O N+ C H H C O N C H H C O– 18 10/21/13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Ty r f-Met Pro U A C P-site E-site A-site A U G G G C A U G C C G U A C G A A G A U U A C U A G G A U 5′ 3′ Ribosome moves along mRNA. Elongation The ribosome advances a distance of one codon. The tRNA that occupied the P-site exits through the E-site and the tRNA that was in the A-site occupies the P-site. A tRNA that recognizes the next codon quickly fills the empty A-site. f-Met Gl u Pro Tyr The ribosome continues advancing down the mRNA in the 5′ to 3′ direction, moving one codon at a time. C G G C U U A U G A U G C C G U A C G A A G A U U A C U A G G A U 5′ 3′ (b) f-Met Pro Tyr Glu Asp Tyr C U A A U G A U G C C G U A C G A A G A U U A C U A G G A U 5′ 3′ Termination Translation continues until a stop codon is reached, signaling the end of the process. No tRNA molecules recognize a stop codon. The components dissemble, releasing the newly formed polypeptide. Pro f-Met Tyr Glu Asp Tyr (c) Both processes occur at the same time in bacteria…why?? 19 10/21/13 Eukaryotic cells differ in transcription and translation • Ribosomes are different size • 5 end of mRNA has cap (methylated guanine) • 3 end of mRNA has poly A tail • Introns are excised, exons spliced together • Translation is monocystronic Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Eukaryotic transcription Exon Intron Exon Eukaryotic DNA contains introns, which interrupt coding regions (exons). Intron Exon Eukaryotic DNA Transcription generates pre-mRNA (precursor mRNA) that contains introns. A cap and poly A tail are then added. Poly A tail Cap Pre-mRNA Splicing removes introns to create functional mRNA. mRNA mRNA is transported out of the nucleus to be translated In the cytoplasm. 20 10/21/13 Is it important to regulate protein synthesis? • Yes! • Three types of protein regulation – Enyme inhibition (feedback inhibition) – Repression (tryptophan operon) – Induction (lactose operon) Are all genes under regulation? • No! • Genes to produce enzymes for glucose metabolism are constitutive (always made) • Other genes are induced…only made when needed • Other genes are repressed…turned off when not needed 21 10/21/13 Models for transcriptional regulation with repressors Transcriptional regulation by activators 22 10/21/13 Lactose Operon as a model • Used to understand control of gene expression in bacteria • Operon consists of three genes needed to degrade lactose • Repressor gene(codes for repressor protein) outside of operon coding region inhibits transcription unless something else bind to the repressor protein Lactose Operon 23 10/21/13 Diauxic growth curve of E. coli 24 10/21/13 What conditions are needed for the lactose operon to be turned on ? • • • • No glucose Lactose present Increasing levels of cAMP cAMP binds to CAP, then complex binds next to lactose operon promoter at the activator region • RNA polymerase binds to promoter How do organisms adapt to other changes in their environment? • Some organisms turn genes on/off as needed • Some organisms alter gene expression 25 10/21/13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Gene regulation systems in bacteria • Signal transduction – Two component regulatory system Environmental stimulus Sensor protein Response regulator The sensor protein spans the cytoplasmic membrane. The response regulator is a protein inside the cell. P P In response to a specific change in the environment, the sensor phosphorylates a region on its internal portion. The phosphate group is transferred to the response regulator, which can then turn genes on or off, depending on the system. • Signal transduction - Quorum sensing Bacterial cell When few cells are present, the concentration of the signaling molecule is low. Signaling molecule When many cells are present, the signaling molecule reaches a concentration high enough to induce the expression of certain genes. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 26 10/21/13 Gene expression is influenced by natural selection • Random changes enhance survival of some cells in population • Antigenic variation of pathogens • Phase variation – Switching on/off of certain genes 27