Chapter 10 Molecular Biology of the Gene

Transcription

1 hapter 0 Molecular Biology of the ene oweroint Lectures for ampbell Biology: oncepts & onnections, Seventh Edition Reece, aylor, Simon, and Dickey Lecture by Edward J. Zalisko Introduction Viruses infect organisms by binding to receptors on a host s target cell, injecting viral genetic material into the cell, and hijacking the cell s own molecules and organelles to produce new copies of the virus. he host cell is destroyed, and newly replicated viruses are released to continue the infection. Introduction Figure 0.0_ hapter 0: Big Ideas Viruses are not generally considered alive because they are not cellular and cannot reproduce on their own. Because viruses have much less complex structures than cells, they are relatively easy to study at the molecular level. For this reason, viruses are used to study the functions of DN. he Structure of the enetic Material he Flow of enetic Information from DN to RN to rotein DN Replication he enetics of Viruses and Bacteria Figure 0.0_2 HE SRUURE OF HE ENEI MERIL

2 0. SIENIFI DISOVERY: Experiments showed that DN is the genetic material Until the 940s, the case for proteins serving as the genetic material was stronger than the case for DN. roteins are made from 20 different amino acids. DN was known to be made from just four kinds of nucleotides. Studies of bacteria and viruses ushered in the field of molecular biology, the study of heredity at the molecular level, and revealed the role of DN in heredity. 0. SIENIFI DISOVERY: Experiments showed that DN is the genetic material In 928, Frederick riffith discovered that a transforming factor could be transferred into a bacterial cell. He found that when he exposed heat-killed pathogenic bacteria to harmless bacteria, some harmless bacteria were converted to disease-causing bacteria and the disease-causing characteristic was inherited by descendants of the transformed cells. 0. SIENIFI DISOVERY: Experiments showed that DN is the genetic material In 952, lfred Hershey and Martha hase used bacteriophages to show that DN is the genetic material of 2, a virus that infects the bacterium Escherichia coli (E. coli). Bacteriophages (or phages for short) are viruses that infect bacterial cells. hages were labeled with radioactive sulfur to detect proteins or radioactive phosphorus to detect DN. Bacteria were infected with either type of labeled phage to determine which substance was injected into cells and which remained outside the infected cell. 0. SIENIFI DISOVERY: Experiments showed that DN is the genetic material he sulfur-labeled protein stayed with the phages outside the bacterial cell, while the phosphorus-labeled DN was detected inside cells. ells with phosphorus-labeled DN produced new bacteriophages with radioactivity in DN but not in protein. Figure 0. Figure 0.B Head ail DN hage Bacterium Batch : Radioactive protein labeled in yellow Radioactive protein DN Empty protein shell hage DN entrifuge ellet he radioactivity is in the liquid. ail fiber 2 4 Batch 2: Radioactive DN labeled in green Radioactive DN entrifuge ellet he radioactivity is in the pellet. 2

3 Figure DN and RN are polymers of nucleotides phage attaches 2 he phage injects he phage DN directs itself to a bacterial its DN into the the host cell to make cell. bacterium. more phage DN and proteins; new phages assemble. 4 he cell lyses and releases the new phages. DN and RN are nucleic acids. One of the two strands of DN is a DN polynucleotide, a nucleotide polymer (chain). nucleotide is composed of a nitrogenous base, five-carbon sugar, and phosphate group. he nucleotides are joined to one another by a sugar-phosphate backbone. 0.2 DN and RN are polymers of nucleotides Figure 0.2 Each type of DN nucleotide has a different nitrogen-containing base: adenine (), cytosine (), thymine (), and guanine (). DN double helix ovalent bond joining nucleotides DN nucleotide Sugar-phosphate backbone hosphate group Nitrogenous base Sugar hosphate group Sugar (deoxyribose) DN nucleotide Nitrogenous base (can be,,, or ) hymine () wo representations of a DN polynucleotide Figure 0.2B 0.2 DN and RN are olymers of Nucleotides RN (ribonucleic acid) is unlike DN in that it uses the sugar ribose (instead of deoxyribose in DN) and hymine () ytosine () denine () uanine () RN has the nitrogenous base uracil (U) instead of thymine. yrimidines urines

4 Figure 0.2 hosphate group Nitrogenous base (can be,,, or U) Figure 0.2D Uracil denine ytosine Uracil (U) Ribose hosphate uanine Sugar (ribose) 0. SIENIFI DISOVERY: DN is a double-stranded helix Figure 0. In 952, after the Hershey-hase experiment demonstrated that the genetic material was most likely DN, a race was on to describe the structure of DN and explain how the structure and properties of DN can account for its role in heredity. 0. SIENIFI DISOVERY: DN is a double-stranded helix In 95, James D. Watson and Francis rick deduced the secondary structure of DN, using X-ray crystallography data of DN from the work of Rosalind Franklin and Maurice Wilkins and hargaff s observation that in DN, the amount of adenine was equal to the amount of thymine and the amount of guanine was equal to that of cytosine. 0. SIENIFI DISOVERY: DN is a double-stranded helix Watson and rick reported that DN consisted of two polynucleotide strands wrapped into a double helix. he sugar-phosphate backbone is on the outside. he nitrogenous bases are perpendicular to the backbone in the interior. Specific pairs of bases give the helix a uniform shape. pairs with, forming two hydrogen bonds, and pairs with, forming three hydrogen bonds. 4

5 Figure 0. Figure 0.D Hydrogen bond Base pair wist Ribbon model artial chemical structure omputer model 0. SIENIFI DISOVERY: DN is a double-stranded helix In 962, the Nobel rize was awarded to James D. Watson, Francis rick, and Maurice Wilkins. Rosalind Franklin probably would have received the prize as well but for her death from cancer in 958. Nobel rizes are never awarded posthumously. he Watson-rick model gave new meaning to the words genes and chromosomes. he genetic information in a chromosome is encoded in the nucleotide sequence of DN. DN RELIION 0.4 DN replication depends on specific base pairing In their description of the structure of DN, Watson and rick noted that the structure of DN suggests a possible copying mechanism. DN replication follows a semiconservative model. he two DN strands separate. Each strand is used as a pattern to produce a complementary strand, using specific base pairing. Each new DN helix has one old strand with one new strand. Figure 0.4_s parental molecule of DN 5

6 Figure 0.4_s2 Figure 0.4_s parental molecule of DN Free nucleotides he parental strands separate and serve as templates parental molecule of DN Free nucleotides he parental strands separate and serve as templates wo identical daughter molecules of DN are formed Figure 0.4B arental DN molecule 0.5 DN replication proceeds in two directions at many sites simultaneously DN replication begins at the origins of replication where Daughter strand arental strand DN unwinds at the origin to produce a bubble, replication proceeds in both directions from the origin, and replication ends when products from the bubbles merge with each other. Daughter DN molecules 0.5 DN replication proceeds in two directions at many sites simultaneously DN replication occurs in the 5 to direction. Replication is continuous on the to 5 template. Replication is discontinuous on the 5 to template, forming short segments. 0.5 DN replication proceeds in two directions at many sites simultaneously wo key proteins are involved in DN replication.. DN ligase joins small fragments into a continuous chain. 2. DN polymerase adds nucleotides to a growing chain and proofreads and corrects improper base pairings. 6

7 0.5 DN replication proceeds in two directions at many sites simultaneously DN polymerases and DN ligase also repair DN damaged by harmful radiation and toxic chemicals. Figure 0.5 arental DN molecule Origin of replication arental strand Daughter strand DN replication ensures that all the somatic cells in a multicellular organism carry the same genetic information. Bubble wo daughter DN molecules Figure 0.5B 5 end end 5 HO Figure arental DN Replication fork DN polymerase molecule 5 his daughter strand is synthesized continuously his daughter strand is synthesized 5 in pieces 5 OH DN ligase end 5 end Overall direction of replication HE FLOW OF ENEI INFORMION FROM DN O RN O ROEIN 0.6 he DN genotype is expressed as proteins, which provide the molecular basis for phenotypic traits DN specifies traits by dictating protein synthesis. he molecular chain of command is from DN in the nucleus to RN and RN in the cytoplasm to protein. ranscription is the synthesis of RN under the direction of DN. ranslation is the synthesis of proteins under the direction of RN. 7

8 Figure 0.6_s Figure 0.6_s2 DN DN ranscription NULEUS RN NULEUS YOLSM YOLSM Figure 0.6_s DN RN rotein ranscription ranslation NULEUS YOLSM 0.6 he DN genotype is expressed as proteins, which provide the molecular basis for phenotypic traits he connections between genes and proteins he initial one gene one enzyme hypothesis was based on studies of inherited metabolic diseases. he one gene one enzyme hypothesis was expanded to include all proteins. Most recently, the one gene one polypeptide hypothesis recognizes that some proteins are composed of multiple polypeptides. 0.7 enetic information written in codons is translated into amino acid sequences he sequence of nucleotides in DN provides a code for constructing a protein. rotein construction requires a conversion of a nucleotide sequence to an amino acid sequence. ranscription rewrites the DN code into RN, using the same nucleotide language. 0.7 enetic information written in codons is translated into amino acid sequences he flow of information from gene to protein is based on a triplet code: the genetic instructions for the amino acid sequence of a polypeptide chain are written in DN and RN as a series of nonoverlapping threebase words called codons. ranslation involves switching from the nucleotide language to the amino acid language. Each amino acid is specified by a codon. 64 codons are possible. Some amino acids have more than one possible codon. 8

9 Figure 0.7 Figure 0.7_ DN molecule ene DN ene 2 ranscription RN U U U U U U U ene ranslation odon DN ranscription RN U U U U U U U olypeptide mino acid ranslation odon olypeptide mino acid 0.8 he genetic code dictates how codons are translated into amino acids haracteristics of the genetic code hree nucleotides specify one amino acid. 6 codons correspond to amino acids. U codes for methionine and signals the start of transcription. stop codons signal the end of translation. 0.8 he genetic code dictates how codons are translated into amino acids he genetic code is redundant, with more than one codon for some amino acids, unambiguous in that any codon for one amino acid does not code for any other amino acid, nearly universal the genetic code is shared by organisms from the simplest bacteria to the most complex plants and animals, and without punctuation in that codons are adjacent to each other with no gaps in between. Figure 0.8 Second base Figure 0.8B_s Strand to be transcribed DN First base hird base 9

10 Figure 0.8B_s2 Strand to be transcribed Figure 0.8B_s Strand to be transcribed DN DN ranscription ranscription RN U U U U U RN U U U U U ranslation Start codon Stop codon olypeptide Met Lys he Figure ranscription produces genetic messages in the form of RN Overview of transcription n RN molecule is transcribed from a DN template by a process that resembles the synthesis of a DN strand during DN replication. RN nucleotides are linked by the transcription enzyme RN polymerase. Specific sequences of nucleotides along the DN mark where transcription begins and ends. he start transcribing signal is a nucleotide sequence called a promoter. 0.9 ranscription produces genetic messages in the form of RN ranscription begins with initiation, as the RN polymerase attaches to the promoter. During the second phase, elongation, the RN grows longer. s the RN peels away, the DN strands rejoin. Finally, in the third phase, termination, the RN polymerase reaches a sequence of bases in the DN template called a terminator, which signals the end of the gene. he polymerase molecule now detaches from the RN molecule and the gene. Figure 0.9 RN polymerase U Direction of transcription Newly made RN U Free RN nucleotides U emplate strand of DN 0

11 Figure 0.9B romoter DN RN polymerase DN of gene Initiation erminator DN Figure 0.9B_ RN polymerase DN of gene erminator DN 2 Elongation rea shown in Figure 0.9 romoter DN Initiation ermination rowing RN ompleted RN RN polymerase Figure 0.9B_2 Figure 0.9B_ 2 Elongation rea shown in Figure 0.9 ermination rowing RN rowing RN ompleted RN RN polymerase 0.0 Eukaryotic RN is processed before leaving the nucleus as mrn Messenger RN (mrn) encodes amino acid sequences and conveys genetic messages from DN to the translation machinery of the cell, which in prokaryotes, occurs in the same place that mrn is made, but in eukaryotes, mrn must exit the nucleus via nuclear pores to enter the cytoplasm. Eukaryotic mrn has introns, interrupting sequences that separate exons, the coding regions. 0.0 Eukaryotic RN is processed before leaving the nucleus as mrn Eukaryotic mrn undergoes processing before leaving the nucleus. RN splicing removes introns and joins exons to produce a continuous coding sequence. cap and tail of extra nucleotides are added to the ends of the mrn to facilitate the export of the mrn from the nucleus, protect the mrn from attack by cellular enzymes, and help ribosomes bind to the mrn.

12 Figure 0.0 DN RN transcript with cap and tail mrn ap Exon Intron Exon oding sequence Intron Exon ranscription ddition of cap and tail Introns removed Exons spliced together ail NULEUS YOLSM 0. ransfer RN molecules serve as interpreters during translation ransfer RN (trn) molecules function as a language interpreter, converting the genetic message of mrn into the language of proteins. ransfer RN molecules perform this interpreter task by picking up the appropriate amino acid and using a special triplet of bases, called an anticodon, to recognize the appropriate codons in the mrn. Figure 0. mino acid attachment site Figure 0.B trn Enzyme Hydrogen bond RN polynucleotide chain nticodon trn molecule, showing its polynucleotide strand and hydrogen bonding simplified schematic of a trn 0.2 Ribosomes build polypeptides Figure 0.2 rowing polypeptide ranslation occurs on the surface of the ribosome. Ribosomes coordinate the functioning of mrn and trn and, ultimately, the synthesis of polypeptides. trn molecules Large subunit Ribosomes have two subunits: small and large. Each subunit is composed of ribosomal RNs and proteins. Small subunit Ribosomal subunits come together during translation. Ribosomes have binding sites for mrn and trns. mrn 2

13 Figure 0.2B Figure 0.2 trn binding sites rowing polypeptide he next amino acid to be added to the polypeptide Large subunit site site mrn trn Small subunit odons mrn binding site 0. n initiation codon marks the start of an mrn message ranslation can be divided into the same three phases as transcription:. initiation, 2. elongation, and. termination. Initiation brings together mrn, a trn bearing the first amino acid, and the two subunits of a ribosome. 0. n initiation codon marks the start of an mrn message Initiation establishes where translation will begin. Initiation occurs in two steps.. n mrn molecule binds to a small ribosomal subunit and the first trn binds to mrn at the start codon. he start codon reads U and codes for methionine. he first trn has the anticodon U. 2. large ribosomal subunit joins the small subunit, allowing the ribosome to function. he first trn occupies the site, which will hold the growing peptide chain. he site is available to receive the next trn. Figure 0. Figure 0.B Start of genetic message ap Initiator trn mrn Met U site Met U site Large ribosomal subunit U U End ail Start codon Small ribosomal subunit 2

14 0.4 Elongation adds amino acids to the polypeptide chain until a stop codon terminates translation Once initiation is complete, amino acids are added one by one to the first amino acid. Elongation is the addition of amino acids to the polypeptide chain. 0.4 Elongation adds amino acids to the polypeptide chain until a stop codon terminates translation Each cycle of elongation has three steps.. odon recognition: he anticodon of an incoming trn molecule, carrying its amino acid, pairs with the mrn codon in the site of the ribosome. 2. eptide bond formation: he new amino acid is joined to the chain.. ranslocation: trn is released from the site and the ribosome moves trn from the site into the site. 0.4 Elongation adds amino acids to the polypeptide chain until a stop codon terminates translation Figure 0.4_s olypeptide mrn site site odons mino acid nticodon Elongation continues until the termination stage of translation, when odon recognition the ribosome reaches a stop codon, the completed polypeptide is freed from the last trn, and the ribosome splits back into its separate subunits. Figure 0.4_s2 olypeptide site site mino acid Figure 0.4_s olypeptide site site mino acid mrn odons nticodon mrn odons nticodon odon recognition odon recognition 2 eptide bond formation 2 eptide bond formation New peptide bond ranslocation 4

15 Figure 0.4_s4 olypeptide site site mino acid 0.5 Review: he flow of genetic information in the cell is DN RN protein mrn odons nticodon odon recognition ranscription is the synthesis of RN from a DN template. In eukaryotic cells, mrn movement transcription occurs in the nucleus and the mrn must travel from the nucleus to the cytoplasm. Stop codon 2 eptide bond formation New peptide bond ranslocation 0.5 Review: he flow of genetic information in the cell is DN RN protein ranslation can be divided into four steps, all of which occur in the cytoplasm:. amino acid attachment, 2. initiation of polypeptide synthesis,. elongation, and 4. termination. Figure 0.5 ranscription DN mrn ranscription RN polymerase ranslation YOLSM mino acid 2 mino acid attachment Enzyme trn nticodon Initiator trn Large ribosomal Initiation of subunit polypeptide synthesis Start odon Small mrn ribosomal subunit New peptide rowing bond forming polypeptide 4 Elongation mrn odons olypeptide 5 ermination Stop codon 0.6 Mutations can change the meaning of genes 0.6 Mutations can change the meaning of genes mutation is any change in the nucleotide sequence of DN. Mutations can involve large chromosomal regions or just a single nucleotide pair. Mutations within a gene can be divided into two general categories.. Base substitutions involve the replacement of one nucleotide with another. Base substitutions may have no effect at all, producing a silent mutation, change the amino acid coding, producing a missense mutation, which produces a different amino acid, lead to a base substitution that produces an improved protein that enhances the success of the mutant organism and its descendant, or change an amino acid into a stop codon, producing a nonsense mutation. 5

16 0.6 Mutations can change the meaning of genes 0.6 Mutations can change the meaning of genes 2. Mutations can result in deletions or insertions that may alter the reading frame (triplet grouping) of the mrn, so that nucleotides are grouped into different codons, lead to significant changes in amino acid sequence downstream of the mutation, and produce a nonfunctional polypeptide. Mutagenesis is the production of mutations. Mutations can be caused by spontaneous errors that occur during DN replication or recombination or mutagens, which include high-energy radiation such as X-rays and ultraviolet light and chemicals. Figure 0.6 Normal hemoglobin DN Mutant hemoglobin DN Figure 0.6B Normal gene mrn rotein U U U U Met Lys he ly la mrn mrn U Nucleotide substitution U U U U Met Lys he Ser U Deleted la Normal hemoglobin lu Sickle-cell hemoglobin Val Nucleotide deletion U U U U Met Lys Leu la His Inserted Nucleotide insertion U U U U Met Lys Leu la His 0.7 Viral DN may become part of the host chromosome HE ENEIS OF VIRUSES ND BERI virus is essentially genes in a box, an infectious particle consisting of a bit of nucleic acid, wrapped in a protein coat called a capsid, and in some cases, a membrane envelope. Viruses have two types of reproductive cycles.. In the lytic cycle, viral particles are produced using host cell components, the host cell lyses, and viruses are released. 6

17 0.7 Viral DN may become part of the host chromosome 2. In the Lysogenic cycle Viral DN is inserted into the host chromosome by recombination. Viral DN is duplicated along with the host chromosome during each cell division. he inserted phage DN is called a prophage. Most prophage genes are inactive. Environmental signals can cause a switch to the lytic cycle, causing the viral DN to be excised from the bacterial chromosome and leading to the death of the host cell. Figure 0.7_s hage ttaches to cell hage DN Bacterial chromosome 4 he cell lyses, releasing phages he phage injects its DN Lytic cycle hages assemble 2 he phage DN circularizes New phage DN and proteins are synthesized Figure 0.7_s2 hage ttaches to cell hage DN Bacterial chromosome 4 he cell lyses, releasing phages he phage injects its DN 7 Environmental Many cell stress divisions Lytic cycle Lysogenic cycle hages assemble 2 he phage DN 6 he lysogenic bacterium circularizes rophage replicates normally OR New phage DN and 5 hage DN inserts into the bacterial proteins are synthesized chromosome by recombination 0.8 ONNEION: Many viruses cause disease in animals and plants Viruses can cause disease in animals and plants. DN viruses and RN viruses cause disease in animals. typical animal virus has a membranous outer envelope and projecting spikes of glycoprotein. he envelope helps the virus enter and leave the host cell. Many animal viruses have RN rather than DN as their genetic material. hese include viruses that cause the common cold, measles, mumps, polio, and IDS. 0.8 ONNEION: Many viruses cause disease in animals and plants he reproductive cycle of the mumps virus, a typical enveloped RN virus, has seven major steps:. entry of the protein-coated RN into the cell, 2. uncoating the removal of the protein coat,. RN synthesis mrn synthesis using a viral enzyme, 4. protein synthesis mrn is used to make viral proteins, 5. new viral genome production mrn is used as a template to synthesize new viral genomes, 6. assembly the new coat proteins assemble around the new viral RN, and 7. exit the viruses leave the cell by cloaking themselves in the host cell s plasma membrane. 0.8 ONNEION: Many viruses cause disease in animals and plants Some animal viruses, such as herpesviruses, reproduce in the cell nucleus. Most plant viruses are RN viruses. o infect a plant, they must get past the outer protective layer of the plant. Viruses spread from cell to cell through plasmodesmata. Infection can spread to other plants by insects, herbivores, humans, or farming tools. here are no cures for most viral diseases of plants or animals. 7

18 Figure 0.8 lycoprotein spike Figure 0.8_ Viral RN (genome) rotein coat Membranous envelope lycoprotein spike rotein coat lasma membrane of host cell Entry YOLSM Viral RN (genome) Membranous envelope 2 Uncoating 4 Viral RN (genome) rotein synthesis RN synthesis by viral enzyme 5 RN synthesis (other strand) lasma membrane of host cell Entry YOLSM mrn emplate New viral proteins 6 ssembly New viral genome Viral RN (genome) 2 Uncoating RN synthesis by viral enzyme Exit 7 Figure 0.8_2 4 mrn rotein synthesis New viral proteins 6 5 RN synthesis (other strand) emplate ssembly New viral genome 0.9 EVOLUION ONNEION: Emerging viruses threaten human health Viruses that appear suddenly or are new to medical scientists are called emerging viruses. hese include the IDS virus, Ebola virus, West Nile virus, and SRS virus. Exit EVOLUION ONNEION: Emerging viruses threaten human health hree processes contribute to the emergence of viral diseases:. mutation RN viruses mutate rapidly. 2. contact between species viruses from other animals spread to humans.. spread from isolated human populations to larger human populations, often over great distances he IDS virus makes DN on an RN template IDS (acquired immunodeficiency syndrome) is caused by HIV (human immunodeficiency virus). HIV is an RN virus, has two copies of its RN genome, carries molecules of reverse transcriptase, which causes reverse transcription, producing DN from an RN template. 8

19 Figure he IDS virus makes DN on an RN template Envelope lycoprotein rotein coat RN (two identical strands) Reverse transcriptase (two copies) fter HIV RN is uncoated in the cytoplasm of the host cell,. reverse transcriptase makes one DN strand from RN, 2. reverse transcriptase adds a complementary DN strand,. double-stranded viral DN enters the nucleus and integrates into the chromosome, becoming a provirus, 4. the provirus DN is used to produce mrn, 5. the viral mrn is translated to produce viral proteins, and 6. new viral particles are assembled, leave the host cell, and can then infect other cells. Figure 0.20B 0.2 Viroids and prions are formidable pathogens in plants and animals Viral RN DN strand Doublestranded DN Viral RN and proteins 2 6 Reverse transcriptase 5 4 NULEUS YOLSM hromosomal DN RN rovirus DN Some infectious agents are made only of RN or protein. Viroids are small, circular RN molecules that infect plants. Viroids replicate within host cells without producing proteins and interfere with plant growth. rions are infectious proteins that cause degenerative brain diseases in animals. rions appear to be misfolded forms of normal brain proteins, which convert normal protein to misfolded form Bacteria can transfer DN in three ways Viral reproduction allows researchers to learn more about the mechanisms that regulate DN replication and gene expression in living cells. Bacteria are also valuable but for different reasons. Bacterial DN is found in a single, closed loop, chromosome. Bacterial cells divide by replication of the bacterial chromosome and then by binary fission. Because binary fission is an asexual process, bacteria in a colony are genetically identical to the parent cell. 9