gene expression

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© 2011 Pearson Education, Inc. Proteins are the links between genotype and phenotype Gene expression - the process by which DNA directs protein synthesis, includes two stages: transcription and translation

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Figure 17.UN01 DNA RNA Protein central dogma - cells are governed by a cellular chain of command: Gene Expression: Transcription = DNA ‘blueprint’  messenger RNA (mRNA) Translation = mRNA (with tRNA )  polypeptide (protein)

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Figure 17.3 DNA mRNA Ribosome Polypeptide TRANSCRIPTION TRANSLATION TRANSCRIPTION TRANSLATION Polypeptide Ribosome DNA mRNA Pre-mRNA RNA PROCESSING (a) Bacterial cell (b) Eukaryotic cell Nuclear envelope

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DNA template strand TRANSCRIPTION mRNA TRANSLATION Protein Amino acid Codon Trp Phe Gly 5  5  Ser U U U U U 3  3  5  3  G G G G C C T C A A A A A A A T T T T T G G G G C C C G G DNA molecule Gene 1 Gene 2 Gene 3 C C The flow of information from gene to protein is based upon triplet codes called codons

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Cracking the Code 64 codons 61 code for amino acids 3 are “stop” signals to end translation “The genetic code is redundant” = more than one codon may specify the same amino acid © 2011 Pearson Education, Inc. from only 4 nucleotide bases we get 20 amino acids

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

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The Genetic Code is universal……… it is shared by the simplest bacteria to the most complex animals Transplanted Genes from one species can be expressed by a another species © 2011 Pearson Education, Inc. Tobacco plant expressing firefly gene Pig expressing jellyfish gene

Figure 17.UN01:

The first stage of gene expression……. 1 st = Transcription = synthesis of RNA © 2011 Pearson Education, Inc. An RNA strand is synthesized that is complementary to a gene on the DNA template strand RNA synthesis follows the same base-pairing rules as DNA, except that uracil substitutes for thymine three stages of transcription Initiation Elongation Termination

Figure 17.3:

Figure 17.7-1 Promoter RNA polymerase Start point DNA 5  3  Transcription unit 3  5  RNA polymerase - pries the DNA strands apart and hooks together the RNA nucleotides promoter - DNA sequence where RNA polymerase attaches ( Promoters signal the transcriptional start point ) stretch of DNA that is transcribed (gene) = transcription unit

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Figure 17.7-2 Promoter RNA polymerase Start point DNA 5  3  Transcription unit 3  5  Initiation 5  3  3  5  Nontemplate strand of DNA Template strand of DNA RNA transcript Unwound DNA 1

Cracking the Code:

Transcription initiation complex forms 3 DNA Promoter Nontemplate strand 5  3  5  3  5  3  Transcription factors RNA polymerase II Transcription factors 5  3  5  3  5  3  RNA transcript Transcription initiation complex 5  3  TATA box T T T T T T A A A A A A A T Several transcription factors bind to DNA 2 A eukaryotic promoter 1 Start point Template strand A promoter called a TATA box is crucial in forming the initiation complex

Figure 17.5:

Figure 17.7-3 Promoter RNA polymerase Start point DNA 5  3  Transcription unit 3  5  Elongation 5  3  3  5  Nontemplate strand of DNA Template strand of DNA RNA transcript Unwound DNA 2 3  5  3  5  3  Rewound DNA RNA transcript 5  Initiation 1 As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time Nucleotides are added to the growing RNA molecule

The Genetic Code is universal………:

Nontemplate strand of DNA RNA nucleotides RNA polymerase Template strand of DNA 3  3  5  5  5  3  Newly made RNA Direction of transcription A A A A A A A T T T T T T T G G G C C C C C G C C C A A A U U U end Elongation

The first stage of gene expression……. 1st = Transcription = synthesis of RNA:

Figure 17.7-4 Promoter RNA polymerase Start point DNA 5  3  Transcription unit 3  5  Elongation 5  3  3  5  Nontemplate strand of DNA Template strand of DNA RNA transcript Unwound DNA 2 3  5  3  5  3  Rewound DNA RNA transcript 5  Termination 3 3  5  5  Completed RNA transcript Direction of transcription (“downstream”) 5  3  3  Initiation 1

Figure 17.7-1:

DNA mRNA Ribosome Polypeptide TRANSCRIPTION TRANSLATION TRANSCRIPTION TRANSLATION Polypeptide Ribosome DNA mRNA Pre-mRNA RNA PROCESSING Eukaryotic cell Nuclear envelope Eukaryotic cells modify RNA after transcription RNA processing – Enzymes in the nucleus modify pre-mRNA before it is dispatched to the cytoplasm

Figure 17.7-2:

5  Exon Intron Exon 5  Cap Pre-mRNA Codon numbers 1 30 31 104 mRNA 5  Cap 5  Intron Exon 3  UTR Introns cut out and exons spliced together 3  105  146 Poly-A tail Coding segment Poly-A tail UTR 1 146 long stretches of noncoding nucleotides called introns exons nucleotide sequences that are expressed RNA splicing - removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence RNA processing

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Figure 17.12-1 RNA transcript (pre-mRNA) 5  Exon 1 Protein snRNA snRNPs Intron Exon 2 Other proteins RNA splicing is carried out by spliceosomes Spliceosomes = a variety of proteins and several small nuclear ribonucleoproteins ( snRNPs ) that recognize splice sites Ribozymes - catalytic RNA molecules that function as enzymes and can also splice RNA

Figure 17.7-3:

Figure 17.12-3 RNA transcript (pre-mRNA) 5  Exon 1 Protein snRNA snRNPs Intron Exon 2 Other proteins Spliceosome 5  Spliceosome components Cut-out intron mRNA 5  Exon 1 Exon 2

Elongation:

Figure 17.10 Protein-coding segment Polyadenylation signal 5  3  3  5  5  Cap UTR Start codon G P P P Stop codon UTR AAUAAA Poly-A tail AAA AAA … The 5  end receives a (modified nucleotide) cap The 3  end gets a poly-A tail These modifications have several functions…….. facilitate the export of mRNA protect mRNA from hydrolytic enzymes help mRNA attach to ribosome RNA processing

Figure 17.7-4:

The Functional and Evolutionary Importance of Introns Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during splicing ………………. alternative RNA splicing The number of different proteins an organism can produce is much greater than its number of genes © 2011 Pearson Education, Inc.

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POLYPEPTIDE PEPTIDE PET TIDE POP YET LET TIE POLE POT LED OLD Nucleotide sequence of DNA/RNA  Possible genes: POLY PE P T IDE PO LY P EPTIDE PO L YPEPT I D E P OL YPEPTI D E

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Gene DNA Exon 1 Exon 2 Exon 3 Intron Intron Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide Figure 17.13 Proteins often consist of multiple regions called domains Often different exons code for the different domains of a protein Exon shuffling may result in the evolution of new proteins

Figure 17.12-1:

DNA mRNA Ribosome Polypeptide TRANSCRIPTION TRANSLATION TRANSCRIPTION TRANSLATION Polypeptide Ribosome DNA mRNA Pre-mRNA RNA PROCESSING Eukaryotic cell Nuclear envelope mRNA is translated into a protein with the help of transfer RNA ( tRNA ) and Ribosomes The second stage of gene expression……. 2 nd = Translation = RNA-directed synthesis of a polypeptide

Figure 17.12-3:

Figure 17.14 Polypeptide Ribosome Trp Phe Gly tRNA with amino acid attached Amino acids tRNA Anticodon Codons U U U U G G G G C A C C C C G A A A C G C G 5  3  mRNA Translation

Figure 17.10:

Figure 17.15 Amino acid attachment site 3  5  Hydrogen bonds Anticodon Anticodon Anticodon 3  5  Hydrogen bonds Amino acid attachment site 5  3  A A G Each tRNA carries a specific amino acid on one end tRNA has an anticodon on the other end the anticodon pairs with a complementary codon on mRNA

The Functional and Evolutionary Importance of Introns:

Aminoacyl-tRNA synthetase (enzyme) Amino acid P P P Adenosine ATP P P P P P i i i Adenosine tRNA Adenosine P tRNA AMP Computer model Amino acid Aminoacyl-tRNA synthetase Aminoacyl tRNA (“charged tRNA”) Figure 17.16-4 a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNA synthetase

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Figure 17.17b Exit tunnel A site – holds the tRNA carrying the next amino acid to be added to the chain Small subunit Large subunit P A P site – holds the tRNA carrying the growing polypeptide chain mRNA binding site E site – the exit site where discharged tRNAs leave the ribosome E Ribosomes The two ribosomal subunits (large and small) are made of proteins and ribosomal RNA (rRNA)

Figure 17.13:

Figure 17.17c Amino end mRNA E 5  Codons 3  tRNA Growing polypeptide Next amino acid to be added to polypeptide chain Make a correct match between the tRNA anticodon and an mRNA codon

The second stage of gene expression…….:

Figure 17.14 Polypeptide Ribosome Trp Phe Gly tRNA with amino acid attached Amino acids tRNA Anticodon Codons U U U U G G G G C A C C C C G A A A C G C G 5  3  mRNA Translation

Figure 17.14:

Translation = Building a Polypeptide three stages of translation Initiation Elongation Termination All three stages require protein “factors” that aid in the translation process © 2011 Pearson Education, Inc.

Figure 17.15:

Figure 17.18 Initiator tRNA mRNA 5  5  3  Start codon Small ribosomal subunit mRNA binding site 3  Translation initiation complex 5  3  3  U U A A G C P P site i  GTP GDP Met Met Large ribosomal subunit E A 5  1 st - small ribosomal subunit binds with mRNA and an initiator t RNA 2 nd - the small subunit moves along the mRNA until it reaches the start codon (AUG) 3 rd - initiation factors bring in the large subunit and completes the translation initiation complex INITIATION

Figure 17.16-4:

Elongation of the Polypeptide Chain Elongation stage - amino acids are added one by one to the preceding amino acid of the growing chain Each addition involves elongation factors and occurs in 3 steps: codon recognition peptide bond formation translocation © 2011 Pearson Education, Inc.

Figure 17.17b:

Amino end of polypeptide mRNA 5  E A site 3  E GTP GDP  P i P A E P A GTP GDP  P i P A E Ribosome ready for next aminoacyl tRNA P site Figure 17.19-4 codon recognition Form peptide bond translocation ELONGATION

Figure 17.17c:

Release factor Stop codon (UAG, UAA, or UGA) 3  5  a stop codon in the mRNA reaches the A site of the ribosome The A site accepts a release factor TERMINATION

Figure 17.14:

Figure 17.20-3 Release factor Stop codon (UAG, UAA, or UGA) 3  5  3  5  Free polypeptide 2 GTP 5  3  2 GDP  2 i P TERMINATION The release factor causes the addition of a water molecule instead of an amino acid Which releases the polypeptide and the translation complex comes apart

Translation = Building a Polypeptide:

Figure 17.21 Completed polypeptide Incoming ribosomal subunits Start of mRNA (5  end) End of mRNA (3  end) Polyribosome Ribosomes mRNA Growing polypeptides A number of ribosomes can translate a single mRNA simultaneously, forming a polyribosome (or polysome )

Figure 17.18:

During and after synthesis, polypeptide chains spontaneously coil and fold into a three-dimensional shape Proteins may also require post-translational modifications before they can do their job Some polypeptides are activated by enzymes Other polypeptides come together to form the subunits of a protein © 2011 Pearson Education, Inc.

Elongation of the Polypeptide Chain:

Free ribosomes synthesize proteins that function in the cytosol ER bound - ribosomes make proteins that are to be secreted from the cell Ribosomes are identical and can switch from free to bound © 2011 Pearson Education, Inc. Polypeptide synthesis always begins in the cytosol ribosomes

Figure 17.19-4:

Ribosome mRNA Signal peptide SRP 1 SRP receptor protein Translocation complex ER LUMEN 2 3 4 5 6 Signal peptide removed CYTOSOL Protein ER membrane A signal-recognition particle (SRP) binds to the signal peptide The SRP brings the signal peptide and its ribosome to the ER Polypeptides destined for secretion are marked by a signal peptide the polypeptide signals the ribosome to attach to the ER because it is destined for secretion

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Figure 17.26 TRANSCRIPTION DNA RNA polymerase Exon RNA transcript RNA PROCESSING NUCLEUS Intron RNA transcript (pre-mRNA) Poly-A Poly-A Aminoacyl- tRNA synthetase AMINO ACID ACTIVATION Amino acid tRNA 5  Cap Poly-A 3  Growing polypeptide mRNA Aminoacyl (charged) tRNA Anticodon Ribosomal subunits A A E TRANSLATION 5  Cap CYTOPLASM P E Codon Ribosome 5  3 

Figure 17.20-3:

Mutations of one or a few nucleotides can affect protein structure and function Mutations - changes in the genetic material The change of even a single nucleotide in a DNA template strand can lead to the production of an abnormal protein © 2011 Pearson Education, Inc.

Figure 17.21:

Figure 17.23 Wild-type hemoglobin Wild-type hemoglobin DNA 3  3  3  5  5  3  3  5  5  5  5  3  mRNA A A G C T T A A G mRNA Normal hemoglobin Glu Sickle-cell hemoglobin Val A A A U G G T T Sickle-cell hemoglobin Mutant hemoglobin DNA C Point mutations - changes in just one base pair of a gene

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Types of Small-Scale Mutations 2 categories of Point mutations : Nucleotide-pair substitutions (silent, missense, or nonsense) nucleotide-pair insertions or deletions © 2011 Pearson Education, Inc.

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Figure 17.24a Wild type DNA template strand mRNA 5  5  Protein Amino end Stop Carboxyl end 3  3  3  5  Met Lys Phe Gly A instead of G Nucleotide-pair substitution: silent Stop Met Lys Phe Gly U instead of C A A A A A A A A A A T T T T T T T T T T C C C C C C G G G G G G A A A A A G G G U U U U U 5  3  3  5  A A A A A A A A A T T T T T T T T T T C C C C G G G G A A A G A A A A G G G U U U U U T U 3  5  Silent substitution have no effect on the amino acid produced by a codon because of redundancy in the genetic code

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Figure 17.24b Wild type DNA template strand mRNA 5  5  Protein Amino end Stop Carboxyl end 3  3  3  5  Met Lys Phe Gly T instead of C Nucleotide-pair substitution: missense Stop Met Lys Phe Ser A instead of G A A A A A A A A A A T T T T T T T T T T C C C C C C G G G G G G A A A A A G G G U U U U U 5  3  3  5  A A A A A A A A A T T T T T T T T T T C C T C G G G A A G A A A A A G G U U U U U 3  5  A C C G Missense substitutions still code for an amino acid, but not the correct amino acid

Figure 17.26:

Figure 17.24c Wild type DNA template strand mRNA 5  5  Protein Amino end Stop Carboxyl end 3  3  3  5  Met Lys Phe Gly A instead of T Nucleotide-pair substitution: nonsense Met A A A A A A A A A A T T T T T T T T T T C C C C C C G G G G G G A A A A A G G G U U U U U 5  3  3  5  A A A A A A A A T T A T T T T T T T C C C G G G A A G U A A A G G U U U U U 3  5  C C G T instead of C C G T U instead of A G Stop Nonsense substitution change amino acid codon into a stop codon, nearly always leads to a nonfunctional protein

Mutations of one or a few nucleotides can affect protein structure and function:

Insertions and Deletions Insertions and deletions - additions or losses of nucleotide pairs in a gene More often has a disastrous effect on the resulting protein Insertion or deletion of nucleotides may alter the reading frame , producing a frameshift mutation © 2011 Pearson Education, Inc.

Figure 17.23:

Figure 17.24d Wild type DNA template strand mRNA 5  5  Protein Amino end Stop Carboxyl end 3  3  3  5  Met Lys Phe Gly A A A A A A A A A A T T T T T T T T T T C C C C C C G G G G G G A A A A A G G G U U U U U Nucleotide-pair insertion: frameshift causing immediate nonsense Extra A Extra U 5  3  5  3  3  5  Met 1 nucleotide-pair insertion Stop A C A A G T T A T C T A C G T A T A T G T C T G G A T G A A G U A U A U G A U G U U C A T A A G

Types of Small-Scale Mutations:

Figure 17.24e DNA template strand mRNA 5  5  Protein Amino end Stop Carboxyl end 3  3  3  5  Met Lys Phe Gly A A A A A A A A A A T T T T T T T T T T C C C C C C G G G G G G A A A A A G G G U U U U U Nucleotide-pair deletion: frameshift causing extensive missense Wild type missing missing A U A A A T T T C C A T T C C G A A T T T G G A A A T C G G A G A A G U U U C A A G G U 3  5  3  3  5  Met Lys Leu Ala 1 nucleotide-pair deletion 5 

Figure 17.24a:

Figure 17.24f DNA template strand mRNA 5  5  Protein Amino end Stop Carboxyl end 3  3  3  5  Met Lys Phe Gly A A A A A A A A A A T T T T T T T T T T C C C C C C G G G G G G A A A A A G G G U U U U U Nucleotide-pair deletion: no frameshift, but one amino acid missing Wild type A T C A A A A T T C C G T T C missing missing Stop 5  3  3  5  3  5  Met Phe Gly 3 nucleotide-pair deletion A G U C A A G G U U U U T G A A A T T T T C G G A A G

Figure 17.24b:

Mutagens Spontaneous mutations can occur during DNA replication, recombination, or repair Mutagens - physical or chemical agents that cause mutations © 2011 Pearson Education, Inc.

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