Gene Expression Chapter 13

Learning Objective 1 What early evidence indicated that most genes specify the structure of proteins? What early evidence indicated that most genes specify the structure of proteins?

Garrod’s Work Inborn errors of metabolism Inborn errors of metabolism evidence that genes specify proteins evidence that genes specify proteins Alkaptonuria Alkaptonuria rare genetic disease rare genetic disease lacks enzyme to oxidize homogentisic acid lacks enzyme to oxidize homogentisic acid Gene mutation Gene mutation associated with absence of specific enzyme associated with absence of specific enzyme

Alkaptonuria

Fig. 13-1, p. 280 Tyrosine Functional enzyme absent Homogentisic acid Functional enzyme present Disease condition Normal metabolism ALKAPTONURIA Maleylacetoacetate Homogentisic acid excreted in urine; turns black when exposed to air H2OH2O CO 2

Learning Objective 2 Describe Beadle and Tatum’s experiments with Neurospora Describe Beadle and Tatum’s experiments with Neurospora

Beadle and Tatum Exposed Neurospora spores Exposed Neurospora spores to X-rays or ultraviolet radiation to X-rays or ultraviolet radiation induced mutations prevented metabolic production of essential molecules induced mutations prevented metabolic production of essential molecules Each mutant strain Each mutant strain had mutation in only one gene had mutation in only one gene each gene affected only one enzyme each gene affected only one enzyme

Beadle-Tatum Experiments

Fig. 13-2, p. 281 Expose Neurospora spores to UV light or X-rays Fungal growth (mycelium) Each irradiated spore is used to establish culture on complete growth medium (minimal medium plus amino acids, vitamins, etc.) 2 Transfer cells to minimal medium plus vitamins Transfer cells to minimal medium plus amino acids Transfer cells to minimal medium (control) Minimal medium plus arginine Minimal medium plus tryptophan Minimal medium plus lysine Minimal medium plus leucine Minimal medium plus other amino acids 1 3

KEY CONCEPTS Beadle and Tatum demonstrated the relationship between genes and proteins in the 1940s Beadle and Tatum demonstrated the relationship between genes and proteins in the 1940s

Learning Objective 3 How does genetic information in cells flow from DNA to RNA to polypeptide? How does genetic information in cells flow from DNA to RNA to polypeptide?

DNA to Protein Information encoded in DNA Information encoded in DNA codes sequences of amino acids in proteins codes sequences of amino acids in proteins 2-step process: 2-step process: 1. Transcription 2. Translation

Transcription Synthesizes messenger RNA (mRNA) Synthesizes messenger RNA (mRNA) complementary to template DNA strand complementary to template DNA strand specifies amino acid sequences of polypeptide chains specifies amino acid sequences of polypeptide chains

Translation Synthesizes polypeptide chain Synthesizes polypeptide chain specified by mRNA specified by mRNA also requires tRNA and ribosomes also requires tRNA and ribosomes Codon Codon sequence of 3 mRNA nucleotide bases sequence of 3 mRNA nucleotide bases specifies one amino acid specifies one amino acid or a start or stop signal or a start or stop signal

DNA to Protein

Fig. 13-4, p. 283 Nontemplate strand Transcription DNA Template strand mRNA (complementary copy of template DNA strand) Codon 1Codon 2Codon 3Codon 4Codon 5Codon 6 Polypeptide MetThrCysGluCysPhe Translation ‘ ‘ ‘ ‘ ‘ ‘

KEY CONCEPTS Transmission of information in cells is typically from DNA to RNA to polypeptide Transmission of information in cells is typically from DNA to RNA to polypeptide

Learning Objective 4 What is the difference between the structures of DNA and RNA? What is the difference between the structures of DNA and RNA?

RNA RNA nucleotides RNA nucleotides ribose (sugar) ribose (sugar) bases (uracil, adenine, guanine, or cytosine) bases (uracil, adenine, guanine, or cytosine) 3 phosphates 3 phosphates RNA subunits RNA subunits covalently joined by 5′ – 3′ linkages covalently joined by 5′ – 3′ linkages form alternating sugar-phosphate backbone form alternating sugar-phosphate backbone

RNA Structure

Fig. 13-3, p. 282 Uracil Adenine Cytosine Guanine

Learning Objective 5 Why is genetic code said to be redundant and virtually universal? Why is genetic code said to be redundant and virtually universal? How may these features reflect its evolutionary history? How may these features reflect its evolutionary history?

Genetic Code mRNA codons mRNA codons specify a sequence of amino acids specify a sequence of amino acids 64 codons 64 codons 61 code for amino acids 61 code for amino acids 3 codons are stop signals 3 codons are stop signals

Codons

Genetic Code Is redundant Is redundant some amino acids have more than one codon some amino acids have more than one codon Is virtually universal Is virtually universal suggesting all organisms have a common ancestor suggesting all organisms have a common ancestor few minor exceptions to standard code found in all organisms few minor exceptions to standard code found in all organisms

KEY CONCEPTS A sequence of DNA base triplets is transcribed into RNA codons A sequence of DNA base triplets is transcribed into RNA codons

Learning Objective 6 What are the similarities and differences between the processes of transcription and DNA replication? What are the similarities and differences between the processes of transcription and DNA replication?

Enzymes Similar enzymes Similar enzymes RNA polymerases (RNA synthesis) RNA polymerases (RNA synthesis) DNA polymerases (DNA replication) DNA polymerases (DNA replication) Carry out synthesis in 5′ → 3′ direction Carry out synthesis in 5′ → 3′ direction Use nucleotides with 3 phosphate groups Use nucleotides with 3 phosphate groups

Antiparallel Synthesis Strands of DNA are antiparallel Strands of DNA are antiparallel Template DNA strand and complementary RNA strand are antiparallel Template DNA strand and complementary RNA strand are antiparallel DNA template read in 3′ → 5′ direction DNA template read in 3′ → 5′ direction RNA synthesized in 5′ → 3′ direction RNA synthesized in 5′ → 3′ direction

Antiparallel Synthesis

Fig. 13-9, p. 287 mRNA transcript Promoter region RNA polymerase Gene 2 3’ Gene 1Gene 3 mRNA transcript 5’ 3’ 5’ 3’ 5’

Base-Pairing Rules In RNA synthesis and DNA replication In RNA synthesis and DNA replication are the same are the same except uracil is substituted for thymine except uracil is substituted for thymine

Transcription

Fig. 13-7, p. 286 Growing RNA strandTemplateDNA strand 5’ end3’ direction Nucleotide added to growing chain by RNA polymerase 3’end 5’ direction

Learning Objective 7 What features of tRNA are important in decoding genetic information and converting it into “protein language”? What features of tRNA are important in decoding genetic information and converting it into “protein language”?

Transfer RNA (tRNA) “Decoding” molecule in translation “Decoding” molecule in translation Anticodon Anticodon complementary to mRNA codon complementary to mRNA codon specific for 1 amino acid specific for 1 amino acid

tRNA

Fig. 13-6a, p. 285 ’ ’ Loop 3 Hydrogen bonds Loop 1 Loop 2 Anticodon

Fig. 13-6b, p. 285 OH 3’ end Amino acid accepting end P 5’ end Hydrogen bonds Loop 3 Loop 1 Modified nucleotides Loop 2 Anticodon

Fig. 13-6c, p. 285 Amino acid (phenylalanine) Anticodon ‘‘

Transfer RNA (tRNA) tRNA tRNA attaches to specific amino acid attaches to specific amino acid covalently bound by aminoacyl-tRNA synthetase enzymes covalently bound by aminoacyl-tRNA synthetase enzymes

Aminoacyl-tRNA

Fig , p. 289 Phenylalanine AMP+ Aminoacyl-tRNA synthetase + Anticodon Amino acidtRNAAminoacyl-tRNA

Stepped Art Fig , p. 289 AMP+ Phenylalanine Amino acid Aminoacyl-tRNA tRNA + Anticodon Aminoacyl-tRNA synthetase

Learning Objective 8 How do ribosomes function in polypeptide synthesis? How do ribosomes function in polypeptide synthesis?

Ribosomes Bring together all machinery for translation Bring together all machinery for translation Couple tRNAs to mRNA codons Couple tRNAs to mRNA codons Catalyze peptide bonds between amino acids Catalyze peptide bonds between amino acids Translocate mRNA to read next codon Translocate mRNA to read next codon

Ribosomal Subunits Each ribosome is made of Each ribosome is made of 1 large ribosomal subunit 1 large ribosomal subunit 1 small ribosomal subunit 1 small ribosomal subunit Each subunit contains Each subunit contains ribosomal RNA (rRNA) ribosomal RNA (rRNA) many proteins many proteins

Ribosome Structure

Fig a, p. 290 Front view Large subunit E P A Ribosome Small subunit

Fig b, p. 290 Large ribosomal subunit E site P site A site mRNA binding site Small ribosomal subunit

KEY CONCEPTS A sequence of RNA codons is translated into a sequence of amino acids in a polypeptide A sequence of RNA codons is translated into a sequence of amino acids in a polypeptide

Animation: Structure of a Ribosome CLICK TO PLAY

Learning Objective 9 Describe the processes of initiation, elongation, and termination in polypeptide synthesis Describe the processes of initiation, elongation, and termination in polypeptide synthesis

Initiation 1st stage of translation 1st stage of translation Initiation factors Initiation factors bind to small ribosomal subunit bind to small ribosomal subunit which binds to mRNA at start codon (AUG) which binds to mRNA at start codon (AUG) Initiator tRNA Initiator tRNA binds to start codon binds to start codon then binds large ribosomal subunit then binds large ribosomal subunit

Elongation A cyclic process A cyclic process adds amino acids to polypeptide chain adds amino acids to polypeptide chain Proceeds in 5′ → 3′ direction along mRNA Proceeds in 5′ → 3′ direction along mRNA Polypeptide chain grows Polypeptide chain grows from amino end to carboxyl end from amino end to carboxyl end

Termination Final stage of translation Final stage of translation when ribosome reaches stop codon when ribosome reaches stop codon A site binds to release factor A site binds to release factor triggers release of polypeptide chain triggers release of polypeptide chain dissociation of translation complex dissociation of translation complex

Stages of Transcription

RNA polymerase binds to promoter region in DNA Termination sequence Promoter region Direction of transcription RNA transcript Rewinding of DNA Unwinding of DNA RNA transcript RNA polymerase DNA DNA template strand Fig. 13-8, p. 287

Learning Objective 10 What is the functional significance of the structural differences between bacterial and eukaryotic mRNAs? What is the functional significance of the structural differences between bacterial and eukaryotic mRNAs?

Eukaryotes Genes and mRNA molecules Genes and mRNA molecules are more complicated than those of bacteria are more complicated than those of bacteria

Eukaryotic mRNA After transcription After transcription 5′ cap (modified guanosine triphosphate) is added to 5′ end of mRNA molecule 5′ cap (modified guanosine triphosphate) is added to 5′ end of mRNA molecule Poly-A tail (adenine-containing nucleotides) Poly-A tail (adenine-containing nucleotides) may be added at 3′ end of mRNA molecule may be added at 3′ end of mRNA molecule

Posttranscriptional Modification

Fig , p st exon 1st intron 2nd exon 2nd intron 3rd exon mRNA termination sequence Promoter Template DNA strand 7-methylguanosine cap Transcription, capping of 5’ end 5’ end Start codon Stop codon Formation of pre-mRNA Small nuclear ribonucleoprotein complex 1st intron 2nd intron 5’ end –AAA... Poly-A tail 3’ end Processing of pre-mRNA (addition of poly-A tail and removal of introns) 2nd exon 3rd exon –AAA... Poly-A tail 3’ end 5’ end Protein-coding region Mature mRNA in nucleus Nuclear envelope Nuclear pore Cytosol Transport through nuclear envelope to cytosol –AAA... Poly-A tail 3’ end 5’ end Start codon Stop codon Mature mRNA in cytosol 1st exon

Introns and Exons Introns Introns noncoding regions (interrupt exons) noncoding regions (interrupt exons) removed from original pre-mRNA removed from original pre-mRNA Exons Exons coding regions in eukaryotic genes coding regions in eukaryotic genes spliced to produce continuous polypeptide coding sequence spliced to produce continuous polypeptide coding sequence

Learning Objective 11 What is the difference between translation in bacterial and eukaryotic cells? What is the difference between translation in bacterial and eukaryotic cells?

Bacterial Cells Transcription and translation are coupled Transcription and translation are coupled Bacterial ribosomes Bacterial ribosomes bind to 5′ end of growing mRNA bind to 5′ end of growing mRNA initiate translation before message is fully synthesized initiate translation before message is fully synthesized

Bacterial mRNA

Fig , p. 288 Promoter region mRNA termination sequence Transcribed region DNA Upstream leader sequences Downstream trailing sequences Protein-coding sequences Translated region Start codonStop codon mRNA 5 ′ end –OH 3 ′ end Polypeptide

Initiation

Fig a, p. 291 Leader sequence mRNA Small ribosomal subunit Initiation factor Start codon

Fig b, p. 291 fMet Initiator tRNA

Fig c, p. 291 fMet Large ribosomal subunit P site E siteA site Initiation complex

Elongation

Fig , p. 292 tRNA with an amino acid Amino acids GDP GTP E PA EPA Aminoacyl- tRNA binds to codon in A site mRNA Ribosome ready to accept another aminoacyl-tRNA Peptide bond formation Amino end of polypeptide New peptide bond Translocation toward 3 ′ end of mRNA EPAE PA GTP GDP

Termination

Fig a, p. 293 Release factor EPA mRNA Stop codon (UAA, UAG, or UGA)

Fig b, p. 293 Polypeptide chain is released Stop codon (UAA, UAG, or UGA)

Fig c, p. 293 Large ribosomal subunit Release factor A P E mRNA Small ribosomal subunit tRNA

Polyribosome Many ribosomes bound to a single mRNA Many ribosomes bound to a single mRNA

KEY CONCEPTS Prokaryotic and eukaryotic cells differ in the details of transcription and translation Prokaryotic and eukaryotic cells differ in the details of transcription and translation

Learning Objective 12 Describe retroviruses and the enzyme reverse transcriptase Describe retroviruses and the enzyme reverse transcriptase

Retroviruses Synthesize DNA from an RNA template Synthesize DNA from an RNA template HIV-1 (virus that causes AIDS) HIV-1 (virus that causes AIDS) Enzyme reverse transcriptase Enzyme reverse transcriptase reverses flow of genetic information reverses flow of genetic information

Reverse Transcription

Fig a, p. 297 Chromosome DNA in nucleus of host cell Provirus inserted into chromosome DNA DNA provirus DNA replication Digestion of RNA strand RNA /DNA hybrid Reverse transcription RNA virus Viral RNA

Fig b, p. 297 Provirus DNA transcribed Viral mRNA Viral RNA Viral proteins RNA virus 2

Learning Objective 13 Give examples of the different classes of mutations that affect the base sequence of DNA Give examples of the different classes of mutations that affect the base sequence of DNA What effects does each have on the polypeptide produced? What effects does each have on the polypeptide produced?

Base Substitution May alter or destroy protein function May alter or destroy protein function missense mutation missense mutation codon change specifies a different amino acid codon change specifies a different amino acid nonsense mutation nonsense mutation codon becomes a stop codon codon becomes a stop codon May have minimal effects May have minimal effects if amino acid is not altered if amino acid is not altered if codon change specifies a similar amino acid if codon change specifies a similar amino acid

Fig a, p. 299 Normal DNA sequence Normal mRNA sequence Normal protein sequence BASE-SUBSTITUTION MUTATIONS Missense mutation Nonsense mutation (Stop)

Animation: Base-Pair Substitution CLICK TO PLAY

Frameshift Mutations Insertion or deletion of one or two base pairs in a gene Insertion or deletion of one or two base pairs in a gene destroys protein function destroys protein function changes codon sequences downstream from the mutation changes codon sequences downstream from the mutation

Fig b, p. 299 FRAMESHIFT MUTATIONS Deletion causing nonsense Deletion causing altered amino acid sequence Normal DNA sequence Normal mRNA sequence Normal protein sequence (Stop)

Animation: Frameshift Mutation CLICK TO PLAY

Transposons Movable DNA sequences Movable DNA sequences “jump” into the middle of a gene “jump” into the middle of a gene Retrotransposons Retrotransposons replicate by forming RNA intermediate replicate by forming RNA intermediate reverse transcriptase converts to original DNA sequence before jumping into gene reverse transcriptase converts to original DNA sequence before jumping into gene

KEY CONCEPTS Mutations can cause changes in phenotype Mutations can cause changes in phenotype

Animation: Protein Synthesis Summary CLICK TO PLAY