Unit 5 Notes

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University of California - Irvine
Biological Sciences
Diane O' Dowd

Unit 5 DNA = Genetic Material The Search for the Genetic Material: Scientific Inquiry  Two chemical components of chromosomes: DNA and proteins  Mendel and Morgan: key in determining the identity of the genetic material Evidence That DNA Can Transform Bacteria  Transformation- a change in genotype and phenotype due to the assimilation of external DNA by a cell  Found studying two strains of bacterium, one pathogenic and one non-pathogenic Evidence That Viral DNA Can Program Cells  Bacteriophages (phages)- viruses that infect bacteria  Viruses- DNA enclosed by a protective coat often made of protein Additional Evidence That DNA Is the Genetic Material  Erwin Chargaff: different amounts of nucleotides in different DNA = DNA is genetic material o Chargaff’s rules  1. Base composition varies between species  2. Within the species, the number of A and T bases are equal and the number of G and C bases are equal Building a Structural Model of DNA: Scientific Inquiry  Watson and Crick: double helix o Two sugar-phosphate backbones are antiparallel o Purine + purine = too wide Pyrimidine + pyrimidine = too narrow Purine + pyrimidine = width consistent with X-ray data Proteins Work Together in DNA Replication and Repair The Basic Principle: Base Pairing to a Template Strand  When a cell copies a DNA molecule, each strand serves as a template for ordering nucleotides into a new, complementary strand DNA Replication: A Closer Look  Getting started o Origin of replication- the particular site that replication of a DNA molecule begins at o Replicaton fork- a Y-shaped region where the parental strands of DNA are being unwound; at the end of a replication bubble o Proteins that participate in unwinding  Helicases- enzymes that untwist the double helix at the replication forks, separating the two parental strands and making them available as template strands  Single-strand binding proteins- bind to the unpaired DNA strands to keep them from re-pairing after the parental strands separate  Topoisomerase- relieves the stress of the tight strain ahead of the replication fork by breaking, swiveling, and rejoining DNA strands o Enzymes that synthesize DNA cannot initiate the synthesis of a polynucleotide  Initial nucleotide chain that is produced during DNA synthesis is actually a short stretch of RNA, not DNA • RNA chain is the primer and is synthesized by the enzyme primase  Synthesizing a New DNA Strand o DNA polymerases- catalyze the synthesis of new DNA by adding nucleotides to a preexisting chain  DNA polymerase III adds a DNA nucleotide to the RNA primer and then continues adding DNA nucleotides, complementary to the parental DNA template strand, to the growing end of the new DNA strand Genes Specify Proteins Through Transcription/Translation  Gene expression- the process by which DNA directs the synthesis of proteins (or, in some cases, just RNA’s) Basic Principles of Transcription and Translation  Genes provide the instructions for making specific proteins, but a gene does not build a protein directly o Bridge between DNA and protein synthesis is the nucleic acid RNA  Transcription- synthesis of RNA using information in DNA o Nucleic acid information is transcribed from DNA to RNA o Messenger RNA (mRNA)- RNA molecule resulting from the complementary sequence of RNA nucleotides assembled from the template strand  Carries a genetic message from the DNA to the protein-synthesizing machinery of the cell  Translation- the synthesis of a polypeptide using the information in the mRNA o A change in language: cell must translate the nucleotide sequence of an mRNA molecule into the amino acid sequence of a polypeptide o Ribosomes- sites of translation; complex particles that facilitate the orderly linking of amino acids into polypeptide chains o In prokaryotes with no membrane-bound organelles, translation occurs before transcription ends; OPPOSITE of what happens in eukaryotes  Transcription occurs in nucleus  mRNA is transported to cytoplasm, where translation occurs o Primary transcript- the initial RNA transcript from any gene, including those specifying RNA that is not translated into protein (initial mRNA undergoes further processing to become a functional mRNA) The Genetic Code Codons: Triplets of Nucleotides  Flow of information from gene to protein is based on a triplet code  For each gene, only one of the two DNA strands is transcribed o Called template strand- provides the pattern, or template, for the sequence of nucleotides in an RNA transcript  mRNA triplets are called codons, and are always written in the 5’  3’ direction Cracking the Code  61/64 triplets code for amino acids o 3 codons that do not designate amino acids are “stop signals” or termination codons, marking the end of translation o AUG codes for the amino acid methionine (Met) and also functions as a “start signal” or initiation codon  AUG signals the protein-synthesizing machinery to begin translating the mRNA at that location  Polypeptide chains begin with methionine when they are synthesized, but an enzyme may subsequently remove this starter amino acid from the chain  There is redundancy but NO ambiguity o Codons synonymous for a particular amino acid differ only in the third nucleotide base of the triplet  Our ability to extract the intended message from a written language depends on reading the symbols in the correct groupings  reading frame Transcription: DNA-Directed Synthesis of RNA Molecular Components of Transcription  RNA polymerase- pries the two strands of DNA apart and joins together RNA nucleotides complementary to the DNA template strand, thus elongating the RNA polynucleotide o Can only assemble a polynucleotide in its 5’  3’ direction o Don’t need a primer, unlike DNA polymerases  Promoter- the DNA sequence where RNA polymerase attaches and initiates transcription o Direction of transcription = “downstream”; other direction = “upstream”  Transcription unit- the stretch of DNA that is transcribed into an RNA molecule Synthesis of an RNA Transcript  RNA polymerase binding and initiation of transcription o Promoter of a gene includes the transcription start point- the nucleotide where RNA synthesis actually begins o RNA polymerase binds in a precise location and orientation on the promoter, determining where transcription starts and which strand will be used as the template o Transcription factors- a collection of proteins that mediate the binding of RNA polymerase and the initiation of transcription  RNA polymerase II binds to the promoter AFTER transcription factors are attached  Promoter called TATA box helps form the initiation complex at a eukaryotic promoter o Transcription factors attached to promoter DNA + polymerase bound in correct orientation = enzyme unwinds the two DNA strands and starts transcribing the template strand  Elongation of the RNA strand o RNA polymerase moves along the DNA, untwisting the double helix, exposing 10-20 nucleotides at a time for pairing  Adds nucleotides to 3’ end of the growing RNA molecule as it continues o New RNA molecule peels away from DNA template, and DNA double helix reforms  Termination of Transcription o RNA polymerase II transcribes a sequence on the DNA called the polyadenylation signal sequence, which codes for a polyadenylation signal (AAUAAA) in the pre-mRNA  At a point 10-35 nucleotides downstream from the AAUAAA signal, proteins associated with the growing RNA transcript cut it free from the polymerase, releasing the pre-mRNA Eukaryotic cells modify RNA after transcription  RNA processing- both ends of the primary transcript are altered Alteration of mRNA Ends  5’ end is synthesized first  receives a 5’ cap- a modified form of a guanine (G) nucleotide added onto the 5’ end after transcription of the first 20-40 nucleotides o 3’ end is also modified before mRNA leaves the nucleus  Pre-mRNA is released soon after the polyadenylation signal, AAUAAA, is transcribed o At 3’ end, enzyme adds 50-250 adenine nucleotides  called a poly-A tail  Both 5’ and 3’ ends: o Facilitate export of mature mRNA from the nucleus o Help protect the mRNA from degradation by hydrolytic enzymes o Help ribosomes attach to the 5’ end of the mRNA once the mRNA reaches the cytoplasm  Untranslated regions of the 5’ and 3’ ends are called UTR’s o Will not be translated into protein but have other functions, such as ribosome binding Split Genes and RNA Splicing  RNA splicing- the removal of large portions of the RNA molecule that is initially synthesized  The sequence of DNA nucleotides that codes for a eukaryotic polypeptide is usually NOT continuous o Introns- noncoding segments of nucleic acid that lie between coding regions o Extrons- regions that are eventually expressed, usually by being translated into amino acid sequences  RNA polymerase II transcribes both introns and extrons from the DNA, but the mRNA molecule that enters the cytoplasm is an abridged version  Signal for RNA splicing is a short nucleotide sequence at each end of an intron o Small nuclear ribonucleoproteins (snRNP) recognize these splice sites; several different snRNP’s join with additional proteins to form an even larger assembly called a spliceosome  Ribozymes- RNA molecules that function as enzymes o Intron RNA functions as a ribozyme and catalyzes its own excision o 3 properties of RNA enable some RNA molecules to function as enzymes:  1. RNA is single-stranded, so a region of an RNA molecule may base-pair with a complementary region elsewhere in the same molecule  3D structure  2. Some bases in RNA contain functional groups that may participate in catalysis  3. The ability of RNA to hydrogen-bond with other nucleic acid molecules (either RNA or DNA) adds specificity to its catalytic activity • Complementary base pairing between RNA of spliceosome and RNA of primary RNA transcript precisely locates the region where the ribozyme catalyzes splicing  Functional and Evolutionary Importance of Introns o Some introns contain sequences that regulate gene expression/affect gene products o Alternative splicing- type of gene regulation at the RNA-processing level in which different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns  Allows for the number of different protein products an organisms produces to be greater than its number of genes o Proteins have modular architecture consisting of discrete structural and functional regions called domains Eukaryotic Gene Expression is Regulated at Many Stages Differential Gene Expression  Differences between cell types are due not to different genes being present but to differential gene expression- the expression of different genes by cells with the same genome  Figure 18.6 Regulation of Chromatin Structure  Histone modifications o Histone modifications play a direct role in the regulation of gene transcription o Histone acetylation- acetyl groups (-COCH 3 are attached to lysines in histone cells Translation: RNA-directed Synthesis of a Polypeptide Molecular Components of Translation  Cell “reads” a genetic message and builds a polypeptide accordingly o Message = series of codons along an mRNA molecule Translator = tRNA (transfer RNA) - tRNA function: to transfer amino acids from the cytoplasmic pool of amino acids to a growing polypeptide in a ribosome  Structure and Function of Transfer RNA o Each type of tRNA molecule translates a particular mRNA codon into a particular amino acid o One end of tRNA has a specific amino acid; the other is a nucleotide triplet called a anticodon, which base-pairs with a complementary codon on mRNA o tRNA is transcribed from DNA templates  is made in the nucleus and then travels from nucleus to cytoplasm, where translation occurs o tRNA molecule consists of a single RNA strand  can fold back upon itself to form a 3-dimensional structure o Accurate translation requires:  1. A tRNA that binds to an mRNA codon specifying a particular amino acid must carry that amino acid, and no other, to the ribosome - Correct matching up of tRNA and amino acid carried out by aminoacyl-tRNA synthetases - Driven by ATP  2. The pairing of the tRNA anticodon with the appropriate mRNA codon - Ex. The nucleotide base U at the 5’ end of a tRNA anticodon can pair with either A or G in the third position (at the 3’ end) of an mRNA codon - Flexible base pairing at this codon position is called wobble  Ribosomes o Ribosomes facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis o Ribosome consists of a large subunit and a small subunit, each made up of proteins and one or more ribosomal RNA’s (rRNA’s) o Ribosomal subunits are exported to cytoplasm via nuclear pores o rRNA is most abundant type of cellular RNA o Each ribosome has three binding sites for tRNA:  1. P-site (peptidyl-tRNA binding site)- holds the tRNA carrying the growing polypeptide chain  2. A site (aminoacyl-tRNA binding site)- holds the tRNA carrying the next amino acid to be added to the chain  3. Discharged tRNAs leave the ribosome from the E site (exit site) Building a Polypeptide  3 stages: o 1. Initiation o 2. Elongation o 3. Termination  Ribosome Association and Initiation of Translation o Initiation stage brings together mRNA, a tRNA bearing the first amino acid of the polypeptide, and the two subunits of a ribosome o Small ribosomal subunit binds to both mRNA and a specific initiator tRNA, which carries the amino acid methionine o Initiator tRNA hydrogen-bonds to the AUG start codon (signals start of translation – established codon reading frame for the mRNA) o Large ribosomal subunit attaches  completing the translation initiation complex  Proteins required to bring these together are called initiation factors  Uses GTP  Elongation of the Polypeptide Chain o Amino acids are added one by one at the C-terminus of the growing chain  Each addition involves elongation factors o Occurs in 3-step cycle:  1. Codon recognition- anticodon of an incoming aminoacyl tRNA base- pairs with the complementary mRNA codon in the A site  2. Peptide bond formation- rRNA molecule of large ribosomal subunit catalyzes the formation of a peptide bond between the amino group of the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site  3. Translocation- ribosome translocates the tRNA in the A sit to the P site. The mRNA moves along with its bound tRNAs, bringing the next codon to be translated into the A site  Termination of Translation o Elongation continues until stop codon reaches
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