The Molecular Basis of Inheritance

1962: Nobel Prize in Physiology and MedicineJames D.WatsonFrancis H.CrickMaurice H. F.WilkinsWhat about?Rosalind FranklinWatson, J.D. and F.H. Crick, Molecular Structure of Nucleic Acids: A Structure for Deoxynucleic Acids. Nature 171 (1953), p. 738.

The Structure of DNADNA is composed of four nucleotides, each containing: adenine, cytosine, thymine, or guanine.

The amounts of A = T, G = C, and purines = pyrimidines [Chargaffs Rule].

DNA is a double-stranded helix with antiparallel strands [Watson and Crick].

Nucleotides in each strand are linked by 5-3 phosphodiester bonds

Bases on opposite strands are linked by hydrogen bonding: A with T, and G with C.

The Basic Principle: Base Pairing to a Template StrandThe relationship between structure and function is manifest in the double helixSince the two strands of DNA are complementary each strand acts as a template for building a new strand in replication

DNA replicationThe parent molecule unwinds, and two new daughter strands are built based on base-pairing rules

ACTAGTGATC

DNA Replication is Semi-conservativeEach 2-stranded daughter molecule is only half newOne original strand was used as a template to make the new strand

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DNA ReplicationThe copying of DNA is remarkable in its speed and accuracyInvolves unwinding the double helix and synthesizing two new strands.More than a dozen enzymes and other proteins participate in DNA replicationThe replication of a DNA molecule begins at special sites called origins of replication, where the two strands are separated

Origins of ReplicationA eukaryotic chromosome may have hundreds or even thousands of replication origins

Mechanism of DNA ReplicationDNA replication is catalyzed by DNA polymerase which needs an RNA primerRNA primase synthesizes primer on DNA strandDNA polymerase adds nucleotides to the 3 end of the growing strand

Mechanism of DNA ReplicationNucleotides are added by complementary base pairing with the template strandThe substrates, deoxyribonucleoside triphosphates, are hydrolyzed as added, releasing energy for DNA synthesis.

The Mechanism of DNA ReplicationDNA synthesis on the leading strand is continuous The lagging strand grows the same general direction as the leading strand (in the same direction as the Replication Fork). However, DNA is made in the 5-to-3 directionTherefore, DNA synthesis on the lagging strand is discontinuousDNA is added as short fragments (Okasaki fragments) that are subsequently ligated together

DNA polymerase I degrades the RNA primer and replaces it with DNA

The Mechanism of DNA ReplicationMany proteins assist in DNA replication

DNA helicases unwind the double helix, the template strands are stabilized by other proteins

Single-stranded DNA binding proteins make the template available

RNA primase catalyzes the synthesis of short RNA primers, to which nucleotides are added.

DNA polymerase III extends the strand in the 5-to-3 direction

DNA polymerase I degrades the RNA primer and replaces it with DNA

DNA ligase joins the DNA fragments into a continuous daughter strand

Enzymes in DNA replication

Helicase protein binds to DNA sequences called origins and unwinds DNA strands.Replication

DNA polymerase enzyme adds DNA nucleotides to the RNA primer.Replication

DNA polymerase enzyme adds DNA nucleotides to the RNA primer.DNA polymerase proofreads bases added and replaces incorrect nucleotides.Replication

Leading strand synthesis continues in a 5 to 3 direction.Replication

Leading strand synthesis continues in a 5 to 3 direction.Discontinuous synthesis produces 5 to 3 DNA segments called Okazaki fragments. Replication

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5 35 3 3 5 3Overall directionof replication

3Leading strand synthesis continues in a 5 to 3 direction.Discontinuous synthesis produces 5 to 3 DNA segments called Okazaki fragments. Okazaki fragmentReplication

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5 3 5 3 35 3 3 5 5 3

Leading strand synthesis continues in a 5 to 3 direction.Discontinuous synthesis produces 5 to 3 DNA segments called Okazaki fragments. Replication

3 5 3Leading strand synthesis continues in a 5 to 3 direction.Discontinuous synthesis produces 5 to 3 DNA segments called Okazaki fragments. Replication

Exonuclease activity of DNA polymerase I removes RNA primers.Replication

Polymerase activity of DNA polymerase I fills the gaps.Ligase forms bonds between sugar-phosphate backbone.Replication

Replication Fork Overview

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ProofreadingDNA must be faithfully replicatedbut mistakes occurDNA polymerase (DNA pol) inserts the wrong nucleotide base in 1/10,000 basesDNA pol has a proofreading capability and can correct errorsMismatch repair: wrong inserted base can be removedExcision repair: DNA may be damaged by chemicals, radiation, etc. Mechanism to cut out and replace with correct bases

MutationsA mismatching of base pairs, can occur at a rate of 1 per 10,000 bases.DNA polymerase proofreads and repairs accidental mismatched pairs.Chances of a mutation occurring at any one gene is over 1 in 100,000Because the human genome is so large, even at this rate, mutations add up. Each of us probably inherited 3-4 mutations!

Proofreading and Repairing DNADNA polymerases proofread newly made DNA, replacing any incorrect nucleotidesIn mismatch repair of DNA, repair enzymes correct errors in base pairingIn nucleotide excision DNA repair nucleases cut out and replace damaged stretches of DNA

Accuracy of DNA ReplicationThe chromosome of E. coli bacteria contains about 5 million bases pairsCapable of copying this DNA in less than an hourThe 46 chromosomes of a human cell contain about 6 BILLION base pairs of DNA!!Printed one letter (A,C,T,G) at a timewould fill up over 900 volumes of Campbell.Takes a cell a few hours to copy this DNAWith amazing accuracy an average of 1 per billion nucleotides

The Central Dogma of Life.

Protein SynthesisThe information content of DNA is in the form of specific sequences of nucleotides along the DNA strandsThe DNA inherited by an organism leads to specific traits by dictating the synthesis of proteinsThe process by which DNA directs protein synthesis, gene expression includes two stages, called transcription and translation

Transcription and TranslationCells are governed by a cellular chain of commandDNA RNA proteinTranscriptionIs the synthesis of RNA under the direction of DNAProduces messenger RNA (mRNA)TranslationIs the actual synthesis of a polypeptide, which occurs under the direction of mRNAOccurs on ribosomes

Transcription and TranslationIn prokaryotes transcription and translation occur together

Figure 17.3a

Transcription and TranslationIn a eukaryotic cell the nuclear envelope separates transcription from translationExtensive RNA processing occurs in the nucleus

TranscriptionTranscription is the DNA-directed synthesis of RNARNA synthesisIs catalyzed by RNA polymerase, which pries the DNA strands apart and hooks together the RNA nucleotidesFollows the same base-pairing rules as DNA, except that in RNA, uracil substitutes for thymine

RNATable 17.1RNA is single stranded, not double stranded like DNARNA is short, only 1 gene long, where DNA is very long and contains many genesRNA uses the sugar ribose instead of deoxyribose in DNARNA uses the base uracil (U) instead of thymine (T) in DNA.

Synthesis of an RNA TranscriptThe stages of transcription areInitiationElongationTermination

Promoters signal the initiation of RNA synthesisTranscription factors help eukaryotic RNA polymerase recognize promoter sequencesA crucial promoter DNA sequence is called a TATA box.

Synthesis of an RNA Transcript - Initiation

Synthesis of an RNA Transcript - ElongationRNA polymerase synthesizes a single strand of RNA against the DNA template strand (anti-sense strand), adding nucleotides to the 3 end of the RNA chainAs RNA polymerase moves along the DNA it continues to untwist the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides

Specific sequences in the DNA signal termination of transcriptionWhen one of these is encountered by the polymerase, the RNA transcript is released from the DNA and the double helix can zip up again.

Synthesis of an RNA Transcript - Termination

Transcription Overview

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Most eukaryotic mRNAs arent ready to be translated into protein directly after being transcribed from DNA. mRNA requires processing.

Transcription of RNA processing occur in the nucleus. After this, the messenger RNA moves to the cytoplasm for translation.

The cell adds a protective cap to one end, and a tail of As to the other end. These both function to protect the RNA from enzymes that would degrade

Most of the genome consists of non-coding regions called introns

Non-coding regions may have specific chromosomal functions or have regulatory purposes

Introns also allow for alternative RNA splicing

Thus, an RNA copy of a gene is converted into messenger RNA by doing 2 things:

Add protective bases to the ends

Cut out the introns

Post Termination RNA Processing

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Alteration of mRNA EndsEach end of a pre-mRNA molecule is modified in a particular wayThe 5 end receives a modified nucleotide capThe 3 end gets a poly-A tail

RNA Processing - SplicingThe original transcript from the DNA is called pre-mRNA.

It contains transcripts of both introns and exons.

The introns are removed by a process called splicing to produce messenger RNA (mRNA)

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RNA Processing - SplicingRibozymes are catalytic RNA molecules that function as enzymes and can splice RNARNA splicing removes introns and joins exons

Figure 17.10

RNA ProcessingRNA Splicing can also be carried out by spliceosomes

Alternative Splicing (of Exons)How is it possible that there are millions of human antibodies when there are only about 30,000 genes?Alternative splicing refers to the different ways the exons of a gene may be combined, producing different forms of proteins within the same gene-coding regionAlternative pre-mRNA splicing is an important mechanism for regulating gene expression in higher eukaryotes

RNA ProcessingProteins often have a modular architecture consisting of discrete structural and functional regions called domainsIn many cases different exons code for the different domains in a protein

Figure 17.12

TranslationTranslation is the RNA-directed synthesis of a polypeptideTranslation involves mRNARibosomes - Ribosomal RNATransfer RNA Genetic coding - codons

The Genetic CodeGenetic information is encoded as a sequence of nonoverlapping base triplets, or codonsThe gene determines the sequence of bases along the length of an mRNA molecule

The Genetic CodeCodons: 3 base code for the production of a specific amino acid, sequence of three of the four different nucleotides

Since there are 4 bases and 3 positions in each codon, there are 4 x 4 x 4 = 64 possible codons

64 codons but only 20 amino acids, therefore most have more than 1 codon

3 of the 64 codons are used as STOP signals; they are found at the end of every gene and mark the end of the protein

One codon is used as a START signal: it is at the start of every protein

Universal: in all living organisms

The Genetic CodeA codon in messenger RNA is either translated into an amino acid or serves as a translational start/stop signal

Transfer RNAConsists of a single RNA strand that is only about 80 nucleotides longEach carries a specific amino acid on one end and has an anticodon on the other endA special group of enzymes pairs up the proper tRNA molecules with their corresponding amino acids.tRNA brings the amino acids to the ribosomes,

The anticodon is the 3 RNA bases that matches the 3 bases of the codon on the mRNA molecule

Transfer RNA3 dimensional tRNA molecule is roughly L shaped

RibosomesRibosomes facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesisThe 2 ribosomal subunits are constructed of proteins and RNA molecules named ribosomal RNA or rRNA

RibosomeThe ribosome has three binding sites for tRNAThe P siteThe A siteThe E site

Building a Polypeptide

Building a Molecule of tRNAA specific enzyme called an aminoacyl-tRNA synthetase joins each amino acid to the correct tRNA

Figure 17.15Amino acidATPAdenosinePyrophosphateAdenosineAdenosinePhosphatestRNAPPPPPPiPiPiPAMPAminoacyl tRNA(an activatedamino acid)Aminoacyl-tRNAsynthetase (enzyme) Active site binds theamino acid and ATP.

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Building a PolypeptideWe can divide translation into three stagesInitiationElongationTerminationThe AUG start codon is recognized by methionyl-tRNA or MetOnce the start codon has been identified, the ribosome incorporates amino acids into a polypeptide chainRNA is decoded by tRNA (transfer RNA) molecules, which each transport specific amino acids to the growing chainTranslation ends when a stop codon (UAA, UAG, UGA) is reached

Initiation of TranslationThe initiation stage of translation brings together mRNA, tRNA bearing the first amino acid of the polypeptide, and two subunits of a ribosome

Elongation of the Polypeptide ChainIn the elongation stage, amino acids are added one by one to the preceding amino acid

Termination of TranslationThe final step in translation is termination. When the ribosome reaches a STOP codon, there is no corresponding transfer RNA. Instead, a small protein called a release factor attaches to the stop codon.The release factor causes the whole complex to fall apart: messenger RNA, the two ribosome subunits, the new polypeptide.The messenger RNA can be translated many times, to produce many protein copies.

Translation: Initiation mRNA binds to a ribosome, and the transfer RNA corresponding to the START codon binds to this complex. Ribosomes are composed of 2 subunits (large and small), which come together when the messenger RNA attaches during the initiation process.

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Translation: ElongationElongation: the ribosome moves down the messenger RNA, adding new amino acids to the growing polypeptide chain. The ribosome has 2 sites for binding transfer RNA. The first RNA with its attached amino acid binds to the first site, and then the transfer RNA corresponding to the second codon bind to the second site.

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Translation: ElongationThe ribosome then removes the amino acid from the first transfer RNA and attaches it to the second amino acid.At this point, the first transfer RNA is empty: no attached amino acid, and the second transfer RNA has a chain of 2 amino acids attached to it.

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The elongation cycle repeats as the ribosome moves down the messenger RNA, translating it one codon and one amino acid at a time.The process repeats until a STOP codon is reached.

Translation: Termination

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PolyribosomesA number of ribosomes can translate a single mRNA molecule simultaneously forming a polyribosomePolyribosomes enable a cell to make many copies of a polypeptide very quickly

Comparing Gene Expression In Prokaryotes And EukaryotesIn a eukaryotic cell:The nuclear envelope separates transcription from translationExtensive RNA processing occurs in the nucleusProkaryotic cells lack a nuclear envelope, allowing translation to begin while transcription progresses

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A summary of transcription and translation in a eukaryotic cell

Figure 17.26

Post-translationThe new polypeptide is now floating loose in the cytoplasm if translated by a free ribosome. Polypeptides fold spontaneously into their active configuration, and they spontaneously join with other polypeptides to form the final proteins.Often translation is not sufficient to make a functional protein, polypeptide chains are modified after translationSometimes other molecules are also attached to the polypeptides: sugars, lipids, phosphates, etc. All of these have special purposes for protein function.

Targeting Polypeptides to Specific LocationsCompleted proteins are targeted to specific sites in the cellTwo populations of ribosomes are evident in cells: free ribsomes (in the cytosol) and bound ribosomes (attached to the ER)Free ribosomes mostly synthesize proteins that function in the cytosol Bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cellRibosomes are identical and can switch from free to bound

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Polypeptide synthesis always begins in the cytosolSynthesis finishes in the cytosol unless the polypeptide signals the ribosome to attach to the ERPolypeptides destined for the ER or for secretion are marked by a signal peptide A signal-recognition particle (SRP) binds to the signal peptideThe SRP brings the signal peptide and its ribosome to the ER

Targeting Polypeptides to Specific Locations

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Mutation Causes and RateThe natural replication of DNA produces occasional errors. DNA polymerase has an editing mechanism that decreases the rate, but it still existsTypically genes incur base substitutions about once in every 10,000 to 1,000,000 cellsSince we have about 6 billion bases of DNA in each cell, virtually every cell in your body contains several mutations Mutations can be harmful, lethal, helpful, silentHowever, most mutations are neutral: have no effectOnly mutations in cells that become sperm or eggsare passed on to future generationsMutations in other body cells only cause trouble when they cause cancer or related diseases

MutagensMutagens are chemical or physical agents that interact with DNA to cause mutations.Physical agents include high-energy radiation like X-rays and ultraviolet light Chemical mutagens fall into several categories.Chemicals that are base analogues that may be substituted into DNA, but they pair incorrectly during DNA replication.Interference with DNA replication by inserting into DNA and distorting the double helix.Chemical changes in bases that change their pairing properties.Tests are often used as a preliminary screen of chemicals to identify those that may cause cancerMost carcinogens are mutagenic and most mutagens are carcinogenic.

Viral MutagensScientists have recognized a number of tumor viruses that cause cancer in various animals, including humansAbout 15% of human cancers are caused by viral infections that disrupt normal control of cell divisionAll tumor viruses transform cells into cancer cells through the integration of viral nucleic acid into host cell DNA.

Point mutationsPoint mutations involve alterations in the structure or location of a single gene. Generally, only one or a few base pairs are involved.Point mutations can signficantly affect protein structure and functionPoint mutations may be caused by physical damage to the DNA from radiation or chemicals, or may occur spontaneouslyPoint mutations are often caused by mutagens

Point MutationThe change of a single nucleotide in the DNAs template strand leads to the production of an abnormal protein

Types of Point MutationsPoint mutations within a gene can be divided into two general categoriesBase-pair substitutions - is the replacement of one nucleotide and its partner with another pair of nucleotidesBase-pair insertions or deletions - are additions or losses of nucleotide pairs in a gene

Base-Pair SubstitutionsSilent - changes a codon but codes for the same amino acidMissense - substitutions that change a codon for one amino acid into a codon for a different amino acidNonsense -substitutions that change a codon for one amino acid into a stop codon

Insertions and DeletionsAre additions or losses of nucleotide pairs in a geneMay produce frameshift mutations that will change the reading frame of the gene, and alter all codons downstream from the mutation.

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