Overview: The Flow of Genetic Information The information content of DNA is in the form of specific sequences of nucleotides The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins Proteins are the links between genotype and phenotype RNA is the manager Gene expression, the process by which DNA directs RNA and protein synthesis, includes two stages: transcription(RNA) and translation(protein) 1

Evidence from the Study of Metabolic Defects In 1902, British physician Archibald Garrod first suggested that genes dictate phenotypes through enzymes that catalyze specific chemical reactions He thought symptoms of an inherited disease reflect an inability to synthesize a certain enzyme Alkaptonuria or “black nappie disease” 2

Nutritional Mutants in Neurospora: George Beadle and Edward Tatum exposed bread mold to X-rays, creating mutants that were unable to survive on minimal media Using crosses, they and their coworkers identified three classes of arginine-deficient mutants, each lacking a different enzyme necessary for synthesizing arginine They developed a “one gene–one enzyme” hypothesis, which states that each gene dictates production of a specific enzyme 3

Lactose Operon of E. coli: In the 1950s, Jacob and Monod worked with bacterial mutants to dissect gene control circuits They and others developed evidence of a short-lived intermediate between a gene and the protein that it coded for This intermediate was required for protein synthesis Shown to be an RNA molecule-was named messenger RNA 4

Basic Principles of Transcription and Translation RNA is the bridge and the gatekeeper between genes and the proteins for which they code Transcription is the synthesis of RNA using coded information in DNA Transcription produces many classes of RNA Translation is the synthesis of a polypeptide, using information in one class: messenger RNA Ribosomes are the sites of translation 5

It is far more complicated than this in real life The “Central Dogma” is the old-fashioned concept that cells are governed by a cellular chain of command: DNA RNA protein Idea developed in 1960s It is far more complicated than this in real life 6

Overview-steps in gene Expression in Prokaryotes and Eukaryotes Figure 17.3 Overview-steps in gene Expression in Prokaryotes and Eukaryotes mRNA = messenger RNA Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA DNA TRANSCRIPTION mRNA Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information. Ribosome TRANSLATION Ribosome TRANSLATION Polypeptide Polypeptide (a) Bacterial cell (b) Eukaryotic cell

Figure 17.4 DNA template strand 5 DNA 3 A C C A A A C C G A G T molecule T G G T T T G G C T C A Gene 1 5 3 TRANSCRIPTION Gene 2 U G G U U U G G C U C A +mRNA 5 3 Codon TRANSLATION Figure 17.4 The triplet code. Protein Trp Phe Gly Ser Gene 3 Amino acid Codons in an mRNA molecule are read by translation machinery in the 5 to 3 direction

The Genetic Code is a triplet code How are the instructions for assembling amino acids into proteins encoded into DNA? There are 20 amino acids, but there are only four nucleotide bases in DNA Three nucleotides correspond to an amino acid? codon 9

The Genetic Code is Universal Figure 17.6 Expression of genes from different species. (a) Tobacco plant expressing (b) Pig expressing a jellyfish a firefly gene gene

The code is punctuated (start and stop) Second mRNA base U C A G UUU UCU UAU UGU U Phe Tyr Cys UUC UCC UAC UGC C U Ser UUA UCA UAA Stop UGA Stop A Leu UUG UCG UAG Stop UGG Trp G CUU CCU CAU CGU U His CUC CCC CAC CGC C C Leu Pro Arg CUA CCA CAA CGA A Gln CUG CCG CAG CGG G First mRNA base (5 end of codon) Third mRNA base (3 end of codon) AUU ACU AAU AGU U Asn Ser AUC Ile ACC AAC AGC C A Thr Figure 17.5 The codon table for mRNA. AUA ACA AAA AGA A Lys Arg AUG Met or start ACG AAG AGG G GUU GCU GAU GGU U Asp GUC GCC GAC GGC C G Val Ala Gly Gly GUA GCA GAA GGA A Glu GUG GCG GAG GGG G The code is punctuated (start and stop)

The code is redundant!(>1 codon/aa) Second mRNA base U C A G UUU UCU UAU UGU U Phe Tyr Cys UUC UCC UAC UGC C U Ser UUA UCA UAA Stop UGA Stop A Leu UUG UCG UAG Stop UGG Trp G CUU CCU CAU CGU U His CUC CCC CAC CGC C C Leu Pro Arg CUA CCA CAA CGA A Gln CUG CCG CAG CGG G First mRNA base (5 end of codon) Third mRNA base (3 end of codon) AUU ACU AAU AGU U Asn Ser AUC Ile ACC AAC AGC C A Thr Figure 17.5 The codon table for mRNA. AUA ACA AAA AGA A Lys Arg AUG Met or start ACG AAG AGG G GUU GCU GAU GGU U Asp GUC GCC GAC GGC C G Val Ala Gly Gly GUA GCA GAA GGA A Glu GUG GCG GAG GGG G The code is redundant!(>1 codon/aa)

4 Important Characteristics of the Genetic Code Triplet: 5’ to 3’ in mRNA “Universal” Punctuated Redundant 13

Molecular Components of Transcription RNA synthesis is catalyzed by RNA polymerase, which separates the DNA strands apart and links together the RNA nucleotides (condensation reaction) The RNA is complementary to the DNA template strand RNA synthesis follows the same base-pairing rules as DNA, except that uracil substitutes for thymine 14

Nontemplate strand of DNA RNA nucleotides RNA polymerase T C C A A A 3 T 5 U C T 3 end T G U A G A C A U C C A C C A 5 A 3 T Figure 17.9 Transcription elongation. A G G T T 5 Direction of transcription Template strand of DNA Newly made RNA

Nontemplate strand of DNA 5 3 3 5 Template strand of DNA Figure 17.7-4 Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase 1 Initiation Nontemplate strand of DNA 5 3 3 5 Template strand of DNA RNA transcript Unwound DNA 2 Elongation Rewound DNA 5 3 3 3 5 5 Figure 17.7 The stages of transcription: initiation, elongation, and termination. RNA transcript 3 Termination 5 3 3 5 5 3 Completed RNA transcript Direction of transcription (“downstream”)

Eukaryotic complexities Nucleus has 3 types of RNA polymerase: (Rpol I, Rpol II, R pol III) All 3 need a lot of help to initiate RNA synthesis Eukaryotic control signals are very complicated

Transcription factors Promoter Nontemplate strand DNA 5′ T A T A A A A 3′ 1 A eukaryotic promoter 3′ A T A T T T T 5′ TATA box Start point Template strand Transcription factors 5′ 3′ 2 Several transcription factors bind to DNA. 3′ 5′ RNA polymerase II Transcription factors Figure 17.8 The initiation of transcription at a eukaryotic promoter 5′ 3′ 3′ 3 Transcription initiation complex forms. 3′ 5′ 5′ RNA transcript Transcription initiation complex

Transcription Terminology RNA polymerase Template/non-template +/- sense Initiation, Elongation, Termination Upstream/downstream Promoter/terminator Transcription unit Rpol II (for messenger RNA) Transcription factor TATA box

Eukaryotes modify RNA after transcription A newly made RNA is called a primary transcript When a new RNA molecule is first made it is not RTU It has to be changed or modified prior to use Enzymes in the eukaryotic nucleus modify primary transcripts before they are sent to the cytoplasm (RNA processing aka RNA modification) Pre-RNA, mature RNA

RNA molecules are usually modified after transcription Enzymes and regulatory RNAs catalyze changes to the RNA molecule before it is ready to be used. Changes or modifications can be at the ends or in the middle. Changes or modifications can involve a single nucleotide at a time or a group. Modifications help to control gene expression 5’ cap and 3’ poly A tail for mRNA 21

Split Genes and RNA Splicing Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides that lie between coding regions These noncoding regions are called intervening sequences, or introns The other regions are called exons because they are eventually expressed, usually translated into amino acid sequences RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence

Splicing is the most dramatic modification: Many genes are organized into “expressed” sections or exons separated by “unexpressed” sections or introns 5 Exon Intron Exon Intron Exon 3 Pre-mRNA Codon numbers 5 Cap Poly-A tail 130 31104 105 146 Introns cut out and exons spliced together mRNA 5 Cap Poly-A tail 1146 5 UTR 3 UTR Figure 17.11 RNA processing: RNA splicing. Coding segment The exons and introns are transcribed into RNA and then the exons are joined together at the RNA level: Splicing

Diverse splicing mechanisms exist Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites The RNAs of the spliceosome also catalyze the splicing reaction

Small RNAs Spliceosome 5′ Pre-mRNA Exon 2 Exon 1 Intron Spliceosome Figure 17.12 A spliceosome splicing a pre-mRNA Spliceosome components mRNA Cut-out intron 5′ Exon 1 Exon 2

Ribozymes Ribozymes are catalytic RNA molecules that function as enzymes and can splice RNA The discovery of ribozymes rendered obsolete the belief that all biological catalysts were proteins

Three properties of RNA enable it to function as a catalyst It can form a three-dimensional structure because of its ability to base-pair with itself Some bases in RNA contain functional groups that may participate in catalysis RNA may hydrogen-bond with other nucleic acid molecules

RNA secondary structure-illustrations U1 snRNA and snRNP tRNA

The Functional and Evolutionary Importance of Introns Some introns contain sequences that may regulate gene expression Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during splicing This is called alternative RNA splicing Consequently, the number of different proteins an organism can produce is much greater than its number of genes

More than one product from each gene Adds flexibility

Alternate splicing works Because genes and proteins DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription Alternate splicing works Because genes and proteins are made of modules RNA processing Translation Exons = gene modules Domains = protein modules Domain 3 Figure 17.13 Correspondence between exons and protein domains. Domain 2 Domain 1 Polypeptide

RNA has more roles and functions than any other component Ribsosomal RNA (rRNA) Messenger RNA (mRNA) Transfer RNA (tRNA) Catalytic RNA (ribozymes) Structural RNA Regulatory RNA The RNA World Hypothesis Did the first life forms evolve as RNA-based systems? Did DNA and protein evolve later?