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HL TOPIC 7.1, 7.2, 7.3, 7.4 NUCLEIC ACIDS AMD PROTEIN SYNTHESIS • Describe the structure of DNA, including the antiparallel strands, 3’–5’ linkages and hydrogen bonding between purines and pyrimidines. • Outline the structure of nucleosomes • State that nucleosomes help to supercoil chromosomes and help to regulate transcription. • Distinguish between unique or single-copy genes and highly repetitive sequences in nuclear DNA. • State that eukaryotic genes can contain exons and introns. Discovery of genetic material • In 1928 Frederict Griffith made an experiment to understand what is genetic material in the cell. DNA or protein? • After these four steps of experiments, scientists tought that there must be a factor which transforms living R bacteria into S bacteria. This factor should bu genetic material. • In 1949 Avery and Mc Carty copmleted the experiment. Heat-killed S strain Heat-killed S strain Protein Alive R strain DNA alive R strain Mice lived Mice died ** They concluded that DNA fragments of dead S bacteria transform living R bacteria into S bacteria. So, genetic material must be DNA not protein. DISCOVERY OF THE DNA • In 1952, Alfred Hershey and Martha Chase used bacteriophages to show that DNA is the genetic material of T2, a virus that infects the bacterium Escherichia coli (E. coli). • Bacteriophages (or phages for short) are viruses that infect bacterial cells. • Phages were labeled with radioactive sulfur to detect proteins or radioactive phosphorus to detect DNA. • Bacteria were infected with either type of labeled phage to determine which substance was injected into cells and which remained outside the infected cell. • The sulfur-labeled protein stayed with the phages outside the bacterial cell, while the phosphorus-labeled DNA was detected inside cells. • Cells with phosphorus-labeled DNA produced new bacteriophages with radioactivity in DNA but not in protein. Figure 10.1B Phage Empty protein shell Radioactive protein Bacterium Phage DNA DNA Batch 1: Radioactive protein labeled in yellow The radioactivity is in the liquid. Centrifuge Pellet 1 Batch 2: Radioactive DNA labeled in green 2 3 4 Radioactive DNA Centrifuge Pellet The radioactivity is in the pellet. Discovery of viral genetic material • Hershey and Chase used radioactive labeling method. They used radioactive sulfur to label protein capcid and radioactive phosphorus to label DNA. Figure 11.2A DNA double helix (2-nm diameter) Metaphase chromosome Nucleosome (10-nm diameter) Linker “Beads on a string” Histones Supercoil (300-nm diameter) Tight helical fiber (30-nm diameter) 700 nm Nucleosome, junk DNA Nucleosome: coiled DNA double helix around proteins called histone. 30 5 of the human DNA is made of genes that we use, 70 % of the DNA we do not use. This part is called non-sense DNA. Figure 10.5B 3 end 5 end P HO 5 4 3 2 1 2 A T 5 P C P G C P P T 3 end P G P OH 3 4 1 A P 5 end Figure 10.3D_2 Hydrogen bond G T C A A C T G Partial chemical structure Prokaryotic genetic material versus eukaryotic genetic material • Prokaryotic DNA is circular DNA. • There is no protein part in prokaryotic genetic material (pure DNA). • It is found in cytoplasm • There is no intron. • Eukaryotic DNA is not circular (linear) • It is called chromatin which is made of DNA and protein • It is found in nucleus, chloroplast, mitochondria. • There are introns. • Intron : A portion of mRNA, as transcribed from the DNA of a eukaryote, which is removed by enzymes before it is used for protein synthesis. • Exon: The portions of mRNA that remain after the excision of the introns. Replication of DNA • Meselson and Stahl proved semi-conservative replication of DNA. They used density gradient centrifugation method. They replicated E-Coli bacteria in 15N medium and then they transferred the bacteria into 14N medium. They extracted DNA of bacteria and centrifuged them. 7.2 DNA replication proceeds in two directions at many sites simultaneously DNA replication begins at the origins of replication where • DNA 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. © 2012 Pearson Education, Inc. DNA replication proceeds in two directions at many sites simultaneously DNA replication occurs in the 5 to 3 direction. • Replication is continuous on the 3 to 5 template. • Replication is discontinuous on the 5 to 3 template, forming short segments. © 2012 Pearson Education, Inc. DNA replication proceeds in two directions at many sites simultaneously Two key proteins are involved in DNA replication. 1. DNA ligase joins small fragments into a continuous chain. 2. DNA polymerase – adds nucleotides to a growing chain and – proofreads and corrects improper base pairings. Animation: Origins of Replication Animation: Leading Strand Animation: Lagging Strand Animation: DNA Replication Review © 2012 Pearson Education, Inc. DNA replication proceeds in two directions at many sites simultaneously DNA polymerases and DNA ligase also repair DNA damaged by harmful radiation and toxic chemicals. DNA replication ensures that all the somatic cells in a multicellular organism carry the same genetic information. © 2012 Pearson Education, Inc. Figure 10.5A Parental DNA molecule Origin of replication “Bubble” Two daughter DNA molecules Parental strand Daughter strand Figure 10.5C DNA polymerase molecule 5 3 Parental DNA Replication fork 5 3 DNA ligase Overall direction of replication 3 5 This daughter strand is synthesized continuously This daughter strand is 3 synthesized 5 in pieces 7.3-7.4 Transcription and Translation DNA specifies traits by dictating protein synthesis. The molecular chain of command is from • DNA in the nucleus to RNA and • RNA in the cytoplasm to protein. Transcription is the synthesis of RNA under the direction of DNA. Translation is the synthesis of proteins under the direction of RNA. © 2012 Pearson Education, Inc. Figure 10.6A_s3 DNA Transcription RNA NUCLEUS Translation Protein CYTOPLASM One gene one enzyme hypothesis The connections between genes and proteins • The initial one gene–one enzyme hypothesis was based on studies of inherited metabolic diseases. • The 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. © 2012 Pearson Education, Inc. Transcription The sequence of nucleotides in DNA provides a code for constructing a protein. • Protein construction requires a conversion of a nucleotide sequence to an amino acid sequence. • Transcription rewrites the DNA code into RNA, using the same nucleotide “language.” © 2012 Pearson Education, Inc. Genetic Code, Codon • The 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 DNA and RNA as a series of three-base “words” called codons. • Translation 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. © 2012 Pearson Education, Inc. Figure 10.7 DNA molecule Gene 1 Gene 2 Gene 3 DNA A A A C C G G C A A A A Transcription RNA Translation U U U G G C Codon Polypeptide Amino acid C G U U U U Figure 10.7_1 DNA A A A C U U U C G G C A A A A C G U U U Transcription RNA Translation Codon Polypeptide Amino acid G G C U Genetic code Characteristics of the genetic code • Three nucleotides specify one amino acid. – 61 codons correspond to amino acids. – AUG codes for methionine and signals the start of transcription. – 3 “stop” codons signal the end of translation. © 2012 Pearson Education, Inc. The genetic code dictates how codons are translated into amino acids The genetic code’s charecteristics • There are more than one codon for some amino acids, • Any codon for one amino acid does not code for any other amino acid, • The genetic code is shared by organisms from the simplest bacteria to the most complex plants and animals so it is nearly universal. • Codons are adjacent to each other with no gaps in between. © 2012 Pearson Education, Inc. Figure 10.8A Third base First base Second base Figure 10.8B_s3 Strand to be transcribed T A C T T C A A A A T C DNA A T G A A G T T T T A G Transcription RNA A U G A A G U U U U A G Translation Start codon Polypeptide Met Stop codon Lys Phe Figure 10.8C The mice to the left and right are engineered to express a green fluorescence protein obtained from a jelly (jellyfish) Transcription Overview of transcription • An RNA molecule is transcribed from a DNA template by a process that resembles the synthesis of a DNA strand during DNA replication. • RNA nucleotides are linked by the transcription enzyme RNA polymerase. • Specific sequences of nucleotides along the DNA mark where transcription begins and ends. • The “start transcribing” signal is a nucleotide sequence called a promoter. © 2012 Pearson Education, Inc. Transcription produces genetic messages in the form of RNA • Transcription begins with initiation, as the RNA polymerase attaches to the promoter. • During the second phase, elongation, the RNA grows longer. • As the RNA peels away, the DNA strands rejoin. • Finally, in the third phase, termination, the RNA polymerase reaches a sequence of bases in the DNA template called a terminator, which signals the end of the gene. • The polymerase molecule now detaches from the RNA molecule and the gene. Animation: Transcription © 2012 Pearson Education, Inc. Figure 10.9A Free RNA nucleotides RNA polymerase C C A A A U C C A T A G G T Direction of transcription Newly made RNA T Template strand of DNA Figure 10.9B RNA polymerase DNA of gene Terminator DNA Promoter DNA 1 Initiation 2 Elongation Area shown in Figure 10.9A 3 Termination Growing RNA Completed RNA RNA polymerase Eukaryotic RNA is processed before leaving the nucleus as mRNA Messenger RNA (mRNA) • encodes amino acid sequences and • conveys genetic messages from DNA to the translation machinery of the cell, which in – prokaryotes, occurs in the same place that mRNA is made, but in – eukaryotes, mRNA must exit the nucleus via nuclear pores to enter the cytoplasm. • Eukaryotic mRNA has – introns, interrupting sequences that separate – exons, the coding regions. © 2012 Pearson Education, Inc. Eukaryotic RNA is processed before leaving the nucleus as mRNA Eukaryotic mRNA undergoes processing before leaving the nucleus. • RNA splicing removes introns and joins exons to produce a continuous coding sequence. • A cap and tail of extra nucleotides are added to the ends of the mRNA to – facilitate the export of the mRNA from the nucleus, – protect the mRNA from attack by cellular enzymes, and – help ribosomes bind to the mRNA. © 2012 Pearson Education, Inc. Figure 10.10 Exon Intron Exon Intron Exon DNA Cap RNA transcript with cap and tail Transcription Addition of cap and tail Introns removed Tail Exons spliced together mRNA Coding sequence NUCLEUS CYTOPLASM The Role of Transfer RNA Transfer RNA (tRNA) molecules function as a language interpreter, • converting the genetic message of mRNA • into the language of proteins. Transfer RNA 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 mRNA. © 2012 Pearson Education, Inc. Figure 10.11A Amino acid attachment site Hydrogen bond RNA polynucleotide chain Anticodon A tRNA molecule, showing its polynucleotide strand and hydrogen bonding A simplified schematic of a tRNA Translation Translation occurs on the surface of the ribosome. • Ribosomes coordinate the functioning of mRNA and tRNA and, ultimately, the synthesis of polypeptides. • Ribosomes have two subunits: small and large. • Each subunit is composed of ribosomal RNAs and proteins. • Ribosomal subunits come together during translation. • Ribosomes have binding sites for mRNA and tRNAs. © 2012 Pearson Education, Inc. Figure 10.12B tRNA binding sites Large subunit P A site site Small subunit mRNA binding site Figure 10.12C The next amino acid to be added to the polypeptide Growing polypeptide mRNA tRNA Codons Translation Translation can be divided into the same three phases as transcription: 1. initiation, 2. elongation, and 3. termination. • Initiation brings together • mRNA, • a tRNA bearing the first amino acid, and • the two subunits of a ribosome. © 2012 Pearson Education, Inc. Translation Initiation establishes where translation will begin. Initiation occurs in two steps. 1. An mRNA molecule binds to a small ribosomal subunit and the first tRNA binds to mRNA at the start codon. – The start codon reads AUG and codes for methionine. – The first tRNA has the anticodon UAC. 2. A large ribosomal subunit joins the small subunit, allowing the ribosome to function. – The first tRNA occupies the P site, which will hold the growing peptide chain. – The A site is available to receive the next tRNA. © 2012 Pearson Education, Inc. Figure 10.13A Start of genetic message Cap End Tail Figure 10.13B Large ribosomal subunit Initiator tRNA P site mRNA U A C A U G Start codon 1 Small ribosomal subunit 2 A site U A C A U G 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. Each cycle of elongation has three steps. 1. Codon recognition: The anticodon of an incoming tRNA molecule, carrying its amino acid, pairs with the mRNA codon in the A site of the ribosome. 2. Peptide bond formation: The new amino acid is joined to the chain. 3. Translocation: tRNA is released from the P site and the ribosome moves tRNA from the A site into the P site. © 2012 Pearson Education, Inc. Translation Elongation continues until the termination stage of translation, when • the ribosome reaches a stop codon, • the completed polypeptide is freed from the last tRNA, and • the ribosome splits back into its separate subunits. Animation: Translation © 2012 Pearson Education, Inc. Figure 10.14_s4 Polypeptide P site mRNA Amino acid A site Anticodon Codons 1 Codon recognition mRNA movement Stop codon 2 New peptide bond 3 Translocation Peptide bond formation CONTROL OF GENE EXPRESSION Gene regulation is the turning on and off of genes. Gene expression is the overall process of information flow from genes to proteins. The control of gene expression allows cells to produce specific kinds of proteins when and where they are needed. Our earlier understanding of gene control came from the study of E. coli. A cluster of genes with related functions, along with the control sequences, is called an operon. With few exceptions, operons only exist in prokaryotes. © 2012 Pearson Education, Inc. REGULATION OF GENE IN PROKARYOTES When an E. coli encounters lactose, all the enzymes needed for its metabolism are made at once using the lactose operon. The lactose (lac) operon includes 1. three adjacent lactose-utilization genes, 2. a promoter sequence where RNA polymerase binds and initiates transcription of all three lactose genes, and 3. an operator sequence where a repressor can bind and block RNA polymerase action. © 2012 Pearson Education, Inc. REGULATION OF GENE IN PROKARYOTES Regulation of the lac operon • A regulatory gene, located outside the operon, codes for a repressor protein. • In the absence of lactose, the repressor binds to the operator and prevents RNA polymerase action. • Lactose inactivates the repressor, so – the operator is unblocked, – RNA polymerase can bind to the promoter, and – all three genes of the operon are transcribed. © 2012 Pearson Education, Inc. Figure 11.1B Operon turned off (lactose is absent): OPERON Regulatory gene Promoter Operator Lactose-utilization genes DNA RNA polymerase cannot attach to the promoter mRNA Protein Active repressor Operon turned on (lactose inactivates the repressor): DNA RNA polymerase is bound to the promoter mRNA Translation Protein Lactose Inactive repressor Enzymes for lactose utilization Figure 11.1B_1 Operon turned off (lactose is absent): OPERON Regulatory Promoter Operator gene Lactose-utilization genes DNA RNA polymerase cannot attach to the promoter mRNA Protein Active repressor Figure 11.1B_2 Operon turned on (lactose inactivates the repressor): DNA RNA polymerase is bound to the promoter mRNA Translation Protein Lactose Inactive repressor Enzymes for lactose utilization Environmental changes and regulation of genes There are two types of repressor-controlled operons. • In the lac operon, the repressor is – active when alone and – inactive when bound to lactose. • In the trp bacterial operon, the repressor is – inactive when alone and – active when bound to the amino acid tryptophan (Trp). © 2012 Pearson Education, Inc. Figure 11.1C trp operon lac operon Promoter Operator Gene DNA Active repressor Active repressor Inactive repressor Lactose Inactive repressor Tryptophan Environmental changes and regulation of genes Another type of operon control involves activators, proteins that turn operons on by • binding to DNA and • making it easier for RNA polymerase to bind to the promoter. Activators help control a wide variety of operons. © 2012 Pearson Education, Inc. Chromosome structure and chemical modifications can affect gene expression Differentiation • involves cell specialization, in structure and function, and • is controlled by turning specific sets of genes on or off. Almost all of the cells in an organism contain an identical genome. The differences between cell types are • not due to the presence of different genes but instead • due to selective gene expression. © 2012 Pearson Education, Inc. Chromosome structure and chemical modifications can affect gene expression Eukaryotic chromosomes undergo multiple levels of folding and coiling, called DNA packing. • Nucleosomes are formed when DNA is wrapped around histone proteins. – This packaging gives a “beads on a string” appearance. – Each nucleosome bead includes DNA plus eight histones. – Stretches of DNA, called linkers, join consecutive nucleosomes. • At the next level of packing, the beaded string is wrapped into a tight helical fiber. • This fiber coils further into a thick supercoil. • Looping and folding can further compact the DNA. © 2012 Pearson Education, Inc. Chromosome structure and chemical modifications can affect gene expression DNA packing can prevent gene expression by preventing RNA polymerase and other transcription proteins from contacting the DNA. Cells seem to use higher levels of packing for longterm inactivation of genes. Highly compacted chromatin, found in varying regions of interphase chromosomes, is generally not expressed at all. Animation: DNA Packing © 2012 Pearson Education, Inc. Figure 11.3 Enhancers Promoter Gene DNA Activator proteins Transcription factors Other proteins RNA polymerase Bending of DNA Transcription Complex assemblies of proteins control eukaryotic transcription Silencers are repressor proteins that • may bind to DNA sequences and • inhibit transcription. Coordinated gene expression in eukaryotes often depends on the association of a specific combination of control elements with every gene of a particular metabolic pathway. © 2012 Pearson Education, Inc. Review: Multiple mechanisms regulate gene expression in eukaryotes These controls points include: 1. chromosome changes and DNA unpacking, 2. control of transcription, 3. control of RNA processing including the – addition of a cap and tail and – splicing, 4. flow through the nuclear envelope, 5. breakdown of mRNA, 6. control of translation, and 7. control after translation including – cleavage/modification/activation of proteins and – breakdown of protein. © 2012 Pearson Education, Inc.