Chapter 12 Molecular Biology Of the Gene

One gene differs from another only by the sequence of the nucleotide bases in DNA How does base sequence determine the uniqueness of a species or of individual traits between members of a species? DNA specifies proteins which make unique structures that make up all the characteristics of an organism DNA’s sequence of nucleotides  sequence of amino acids  specific enzymes  structures

In 1900’s, scientists knew the genetic material: Must store information about development, structure, and metabolic activities of a cell Must be stable, so it could be replicated in cell division and passed on Must be able to undergo rare changes called mutations to provide variability required for evolution In the1920’s, Frederick Griffith was working on a vaccine against Streptococcus pneumoniae Notices some colonies were shiny and smooth Other colonies had a rough appearance

Smooth colony individuals had a capsule, but those of the rough colony did not S strain caused death when injected into mice R strain did not Heat-killed S strain mixed with living R strain caused death in mice when injected and when they were isolated from the mice had capsules

Fig. 12.1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Oswald Avery worked on whether genetic material was protein or DNA Subjected materials to proteinases and a capsule was still produced Subjected materials to DNase and it was not DNA was shown to be the genetic material DNA structure: Contains four nitrogenous bases -Two purines- adenine (A) and guanine (G) that were double ringed -Two pyrimidines- thymine (T) and cytosine (C) that were single ringed

Percentage of each type of nucleotide differs from species to species Within a species, DNA has a constancy of bases % of A always equals % of T and % of G always equally % of C. These relationships are called Chargaff’s rules

Fig. 12.3 NH2 C CH3 adenine (A) C thymine (T) HN C N C N CH Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. NH2 C CH3 adenine (A) C thymine (T) HN C N C N CH nitrogen-containing base O C CH HC C N N N O O HO P O 5 CH2 O HO P O 5 CH2 O O C 4 H H C O C H H C 1 4 1 sugar = deoxyribose H C C H H C C H NH2 3 2 O 3 2 OH H OH H C guanine (G) C cytosine (C) C N N CH HN CH C O C CH H2N C N N O N O phosphate HO P O 5 CH2 O 5 HO P O CH2 O O C H H C O C 4 1 H H C 4 1 H C C H H C H C2 3 2 3 a. Purine nucleotides OH H b. Pyrimidine nucleotides OH H DNA Composition in Various Species (%) Species A T G C Homo sapiens (human) 31.0 31.5 19.1 18.4 Drosophila melanogaster (fruit fly) 27.3 27.6 22.5 22.5 Zea mays (corn) 25.6 25.3 24.5 24.6 Neurospora crassa (fungus) 23.0 23.3 27.1 26.6 Escherichia coli (bacterium) 24.6 24.3 25.5 25.6 Bacillus subtilis (bacterium) 28.4 29.0 21.0 21.6 c. Chargaff’s data

Each human chromosome usually contains about 140 million base pairs Because any of the four nucleotides can be present at each position, the total possible nucleotide sequences is 4 to the 140th x 10 to the 6th or 4 to the 140,000,000th Rosalind Franklin’s work with x-ray diffraction determined that DNA was a double helix

Fig. 12.4 Rosalind Franklin diffraction pattern diffracted X-rays a. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Rosalind Franklin diffraction pattern diffracted X-rays a. X-ray beam Crystalline DNA b. c. © Photo Researchers, Inc.; 12.4c: © Science Source/Photo Researchers, Inc.

Watson and Crick constructed a model of DNA and received the 1962 Nobel prize Polymers of nucleotides in a double helix form Sugar-phosphate back bones on outside and paired bases inside Two DNA strands are antiparallel, meaning that the sugar-phosphate groups of each strand are oriented in opposite directions 5’ end of one strand is paired to the 3’ end of the other strand Complementary base pairing means a purine always bonds to a pyrimidine

They have an antiparallel arrangement to insure that bases are oriented properly so can interact This is the only model having molecular width revealed by Franklin’s x-ray diffraction pattern

Fig. 12.5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3.4 nm 0.34 nm 2 nm b. d. d. C a. sugar-phosphate backbone G T 3 end P 5 end 5 G C 2 3 A 4 S 1 P 1 S 4 A 3 2 5 T P P c. G C P Complementary base pairing C G P a: © Kenneth Eward/Photo Researchers, Inc.; d: © A. Barrington Brown/Photo Researchers, Inc.

Term DNA replication refers to process of copying a DNA molecule A template is a mold used to produce a shape complementary to itself During DNA replication, each DNA strand serves as a template for a new strand in a daughter molecule DNA replication is semiconservative replication because each daughter DNA double helix contains an old strand from parential DNA double helix and a new strand

daughter DNA double helix Fig. 12.6 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. G C G C G A T A region of parental DNA double helix T A C G A T A G C G C G A region of replication: new nucleotides are pairing with those of parental strands C G C G A T A T T A T A G C T C A T A G A T A A A T G A G T G T region of completed replication A C C C A G C G new strand old strand A daughter DNA double helix old strand new strand daughter DNA double helix

Steps in replication: 1) Unwinding of parental DNA is caused by the breaking of weak hydrogen bonds between paired bases Enzyme called helicase necessary to unwind the molecule 2) Complementary base pairing occurs when new complementary nucleotides, always in the nucleus, are paired 3) Joining finishes replication by joining the complementary nucleotides to form new strands Each new daughter DNA molecule contains an old strand and a new strand

Steps 2 and 3 are done with the enzyme complex called DNA polymerase DNA replication must occur before a cell can divide Cancer is characterized by rapid cell division Sometimes treated with chemotherapeutic drugs that are analogs to one of four nucleotides Analogs have similar but not identical structure They cause replication to stop and cells to die Bacteria have a single circular loop of DNA that must be replicated before the cell divides Process begins at origin of replication site

The strands are separated, unwound, and DNA polymerase binds to each side and begins copying The two DNA polymerases meet at a termination region, then the chromosomes separate Bacteria cells require about 40 minutes to replicate, but bacterial cells can divide every 20 minutes So replication can begin even before previous round is complete In eukaryotes, DNA replication begins at numerous origins of replication along the length of the chromosome

Replication fork is the V shape of strands formed when replication bubbles spread bidirectionly until they meet Chromosomes are long and linear and replicate at about 500-5,000 base pairs per minute Because there are many individual origins of replication, the diploid DNA in humans of over 6 billion base pairs takes some hours In linear chromosomes, DNA polymerase cannot replicate to the ends Ends of chromosomes composed of telomeres which are short DNA sequences that are repeated over and over

Telomeres are added back by the enzyme telomerase Eventually telomeres are lost and the cell cannot replicate In stem cells, this process preserves the ends of chromosomes and prevents the loss of DNA after successive rounds of replication DNA polymerase is very accurate and makes a mistake approximately once per 100,000 base pairs, but has proof reading capability so overall error rate is one in 100 million base pairs

Fig. 12.7 origin replication is complete replication is occurring in Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. origin replication is complete replication is occurring in two directions a. Replication in prokaryotes replication fork replication bubble parental strand new DNA duplexes daughter strand b. Replication in eukaryotes

RNA is a polymer composed of nucleotides Contains sugar ribose and bases adenine (A) cytosine, (C), guanine (G),and uracil (U) which replaces the thymine in DNA Single stranded and does not form a helix RNA comes in three major classes: Messenger RNA (mRNA) takes message from DNA in nucleus to ribosomes in cytoplasm Transfer RNA (tRNA) transfers amino acids to the ribosomes Ribosomal RNA (rRNA) along with ribosomal proteins, make up the ribosomes where polypeptides are synthesized

Two major steps in synthesizing a protein from information in the DNA Transcription where one of the DNA strands acts as a template to make messenger RNA Translation where the messenger RNA directs the sequence of amino acids into a polypeptide Sequence of nucleotides in DNA to mRNA specify the order of amino acids in polypeptide Genetic code: Codon is a triplet code where three nucleotides code for one of the twenty amino acids Code is degenerate meaning most amino acids have more than one codon

Code is unambiguous meaning each triplet codon has only one meaning Code has one start signal (AUG) and three stop signals (UAA) (UGA) and (UAG) Code is universal

Fig. 12.10 First Base Second Base Third Base U C A G UUU phenylalanine Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. First Base Second Base Third Base U C A G UUU phenylalanine UCU serine UAU tyrosine UGU cysteine U UUC phenylalanine UCC serine UAC tyrosine UGU cysteine C U UUA leucine UCA serine UAA stop UGA stop A UUG leucine UCG serine UAG stop UGG tryptophan G CUU leucine CCU proline CAU histidine CGU arginine U CUC leucine CCC proline CAC histidine CGC arginine C C CUA leucine CCA proline CAA glutamine CGA arginine A CUG leucine CCG proline CAG glutamine CGG arginine G AUU isoleucine ACU threonine AAU asparagine AGU serine U AUC isoleucine ACC threonine AAC asparagine AGC serine C A AUA isoleucine ACA threonine AAA lysine AGA arginine A AUG (start) methionine ACG threonine AAG lysine AGG arginine G GUU valine GCU alanine GAU aspartate GGU glycine U GUC valine GCC alanine GAC aspartate GGC glycine C G GUA valine GCA alanine GAA glutamate GGA glycine A GUG valine GCG alanine GAG glutamate GGG glycine G

Messenger RNA has a sequence of bases complementary to a portion of one DNA strand When a gene is transcribed, a segment of DNA helix unwinds and unzips Rna nucleotides pair with complementary DNA nucleotides; this is known as the template strand RNA polymerase joins the nucleotides together in the 5’  3’ direction or adds a nucleotide to the 3’ end of polymer under construction Transcription begins when RNA polymerase attaches to promoter in DNA

Promoter defines the start of transcription, the direction of transcription, and the strand to be transcribed Initiation of transcription is the binding of RNA polymerase to promoter Elongation of mRNA continues until RNA polymerase comes to DNA stop codon sequence Causes release of mRNA now called mRNA transcript RNA transcript is called pre-mRNA and is modified before leaving the nucleus Receives a cap at 5’ end and a tail at 3’ end

Cap is modified guanine (G) nucleotide which tells ribosome where to attach when translation begins Tail consists of 150-200 adenine (A) nucleotides This poly-A tail helps transport of mRNA out of nucleus and inhibits degration of mRNA by hydrolytic enzymes Pre-mRNA is composed of exons and introns Exons are segments that will be expressed Introns are segments in between exons that will not be expressed During pre-mRNA splicing, introns are removed

In prokaryotes, introns splice themselves out In eukaryotes, DNA splicing is done by spliceosomes which contain small nuclear RNA (snRNA) Spliceosomes use ribozymes whose catalytic activity works like enzymes that are made of protein Presence of introns allows a cell to pick and chose which exons will go into a particular mRNA What is an exon in one mRNA could be an intron in another mRNA Called alternate mRNA splicing

Some introns give rise to micro RNA (miRNA) which are involved in regulating the translation of mRNA Introns may also encourage crossing-over during meiosis, and permit exon shuffling which can play a role in evolution of new genes

Fig. 12.13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. exon exon exon DNA intron intron transcription exon exon exon pre-mRNA 5 intron intron 3 exon exon exon 5 3 cap intron intron poly-A tail spliceosome exon exon exon 5 3 cap poly-A tail pre-mRNA splicing intron RNA mRNA 5 3 cap poly-A tail nuclear pore in nuclear envelope nucleus cytoplasm

Translation takes place in the cytoplasm of eukaryotic cells Transfer RNA (tRNA) molecules transfer amino acids to the ribosomes tRNA is a single stranded nucleic acid that doubles back on itself to create regions where complementary bases hydrogen bond to one another There is at least one tRNA molecule for each of the 20 amino acids Amino acids bind to 3’ end of tRNA The opposite end contains an anticodon that is complementary to a specific mRNA codon

Fig. 12.14 amino acid leucine 3 5 Hydrogen bonding amino acid end Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. amino acid leucine 3 5 Hydrogen bonding amino acid end anticodon G A A anticodon end C A G U C C U U C C U C mRN A 5 3 codon a. b.

In eukaryotes, ribosomal RNA (rRNA) is produced from a DNA template in the nucleolus of a nucleus Packaged with proteins into two ribosomal subunits one larger than the other They move into the cytoplasm where they combine when translation begins May remain in cytoplasm or attach to endoplasmic reticulum Ribosomes have a binding site for mRNA and three binding sites for tRNA When ribosome moves down a mRNA molecule, the polypeptide increases by one AA at a time

Several ribosomes are often attached to and translating the same mRNA The entire complex is called a polyribosome

Courtesy Alexander Rich Fig. 12.15 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. large subunit 5 3 mRNA tRNA binding sites small subunit a. Structure of a ribosome b. Binding sites of ribosome outgoing tRNA polypeptide incoming tRNA mRNA c. Function of ribosomes d. Polyribosome Courtesy Alexander Rich

Translation requires three steps: 1) Initiation brings all the translation components together Initiation factors (proteins) assemble small ribosome subunit, mRNA, initiator tRNA, and large ribosomal subunit 2) Elongation where polypeptide increases in length one amino acid at a time Requires elongation factors (proteins) for binding tRNA anticodons to mRNA codons 3) Termination of polypeptide occurs at a stop codon

Requires protein called release factor which binds to stop codon and cleaves the polypeptide from the last tRNA

Fig. 12.17 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Met Met asp Met Met Thr peptide bond tRNA Ser Ser Ser Ser Ala Ala Ala C U G Ala Trp peptide Trp U G Trp anticodon Trp Val bond G Val Val Val Asp Asp Asp A U C C A U C A U C U G C A U C U G C U G G U A G A C G U A G A C G U A G A C G U A G A C A C C 3 3 5 5 5 3 5 3 1 A tRNA–amino acid approaches the ribosome and binds at the A site. 2 Two tRNAs can be at a ribosome at one time; the anticodons are paired to the codons. 3 Peptide bond formation attaches the peptide chain to the newly arrived amino acid. 4 The ribosome moves forward; the “empty” tRNA exits from the E site; the next amino acid–tRNA complex is approaching the ribosome. Elongation

Fig. 12.18 release factor stop codon Termination 3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. release factor Asp Ala T rp V al U U A A Glu U G A stop codon 3 The ribosome comes to a stop codon on the mRNA. A release factor binds to the site. U U C G A A A U G 3 5 The release factor hydrolyzes the bond between the last tRNA at the P site and the polypeptide, releasing them. The ribosomal subunits dissociate. Termination

Gene has been expressed once its product is made and is operating in the cell Eukaryotic chromosome contains a single double helix DNA molecule, but is composed of more than 50% protein Histones are the large majority and play a primary structural role A human cell contains at least 2 meters of DNA