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Chapter 12 Gene Expression and Regulation Lecture Outlines by Gregory Ahearn, University of North Florida Copyright © 2011 Pearson Education Inc. Chapter 12 At a Glance 12.1 How Is the Information in DNA Used in a Cell? 12.2 How Is the Information in a Gene Transcribed into RNA? 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? 12.4 How Do Mutations Affect Protein Function? 12.5 How Are Genes Regulated? Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.1 How Is the Information in DNA Used in a Cell? The link between DNA and protein – DNA contains the “molecular blueprint” of every cell – Proteins are the construction workers of the cell – Proteins control cell shape, function, reproduction, and synthesis of biomolecules – Therefore, there must be a flow of information from DNA to protein Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.1 How Is the Information in DNA Used in a Cell? Most genes contain the information for the synthesis of a single protein – Experiments on bread molds in the 1940s showed that one gene codes for one protein Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.1 How Is the Information in DNA Used in a Cell? DNA provides instructions for protein synthesis via RNA intermediaries – DNA in eukaryotes is kept in the nucleus – Protein synthesis occurs at ribosomes in the cytoplasm – DNA information must be carried by an intermediary, ribonucleic acid (RNA), from the nucleus to the cytoplasm Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.1 How Is the Information in DNA Used in a Cell? DNA provides instructions for protein synthesis via RNA intermediaries (continued) – RNA differs structurally from DNA in three ways –RNA is usually single-stranded –RNA has the sugar ribose rather than deoxyribose in its backbone –RNA contains the nitrogenous base uracil (U) instead of thymine (T) Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Table 12-1 Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.1 How Is the Information in DNA Used in a Cell? DNA provides instructions for protein synthesis via RNA intermediaries (continued) – There are three types of RNA involved in protein synthesis –Messenger RNA (mRNA) carries DNA gene information to the ribosome –Transfer RNA (tRNA) brings amino acids to the ribosome –Ribosomal RNA (rRNA) is part of the structure of ribosomes Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.1 How Is the Information in DNA Used in a Cell? DNA provides instructions for protein synthesis via RNA intermediaries (continued) – RNA occurs in many other roles besides protein synthesis – RNA is used as the genetic material in some viruses, such as HIV – Enzymatic RNA, called ribozymes, catalyzes various reactions, including the cutting apart of other molecules of RNA – Xist RNA prevents the genetic information in one of the X chromosomes of female mammals from being used – MicroRNA may play a role in regulating development and fighting disease Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Cells Synthesize Three Major Types of RNA That Are Required for Protein Synthesis codons A U G U G C G A G U U A (a) Messenger RNA (mRNA) The base sequence of mRNA carries the information for the amino acid sequence of a protein; groups of these bases, called codons, specify the amino acids catalytic site large subunit 1 2 tRNA/amino acid binding sites small subunit rRNA combines with proteins to form ribosomes; the small subunit binds mRNA; the large subunit binds tRNA and catalyzes peptide bond formation between amino acids during protein synthesis (b) Ribosome: contains ribosomal RNA (rRNA) tyr attached amino acid tRNA anticodon (c) Transfer RNA (tRNA) Biology: Life on Earth, 9e Each tRNA carries a specific amino acid (in this example, tyrosine [tyr]) to a ribosome during protein synthesis; the anticodon of tRNA pairs with a codon of mRNA, ensuring that the correct amino acid is incorporated into the protein Fig. 12-1 Copyright © 2011 Pearson Education Inc. 12.1 How Is the Information in DNA Used in a Cell? DNA provides instructions for protein synthesis via RNA intermediaries (continued) – Messenger RNA carries the code for protein synthesis from DNA to the ribosomes – Ribosomal rRNA and proteins form ribosomes – Ribosomes, the structures that carry out translation, are composed of rRNA and many different proteins – Each ribosome consists of two subunits—one small and one large—that contain various binding and catalytic sites needed for protein synthesis – Transfer tRNA carries amino acids to the ribosomes for addition to the growing protein Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.1 How Is the Information in DNA Used in a Cell? Overview: Genetic information is transcribed into RNA and then translated into protein – DNA directs protein synthesis in a two-step process 1. Information in a DNA gene is copied into RNA in the process of transcription 2. Messenger RNA, together with tRNA, amino acids, and a ribosome, synthesizes a protein in the process of translation of the genetic information contained in the mRNA Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Genetic Information Flows from DNA to RNA to Protein gene DNA (nucleus) (cytoplasm) Transcription of the gene produces an (a) Transcription mRNA with a nucleotide sequence complementary to one messenger RNA of the DNA strands Translation of the mRNA produces a protein molecule with an amino acid sequence determined by the nucleotide sequence in the mRNA (b) Translation ribosome protein Fig. 12-2 Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Table 12-2 Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.1 How Is the Information in DNA Used in a Cell? The genetic code uses three bases to specify an amino acid – The genetic code translates the sequence of bases in nucleic acids into the sequence of amino acids in proteins – Given that there are 20 amino acids but only four bases, statistically, the smallest number of bases that could combine to yield a different sequence for each of the 20 amino acids is three – A two-base code could produce only 16 combinations – The three-base code has the potential to create 64 combinations – Bases in mRNA are read by the ribosome in triplets called codons Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.1 How Is the Information in DNA Used in a Cell? The genetic code uses three bases to specify an amino acid (continued) – Marshall Nirenberg and Heinrich Matthaei cracked the genetic code by creating artificial mRNAs of known sequence and observing what proteins they produced – For example, an mRNA strand composed entirely of uracil (UUUUUUUU…) produced a protein consisting entirely of the amino acid phenylalanine – Therefore, they concluded that the triplet UUU is the codon for phenylalanine Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.1 How Is the Information in DNA Used in a Cell? The genetic code uses three bases to specify an amino acid (continued) – The genetic code is usually written in terms of the base triplets in mRNA (rather than in DNA) – Each codon specifies a unique amino acid in the genetic code – Each mRNA also has a start codon (AUG) and one of three stop codons (UAG, UAA, and UGA) – Each codon species only one specific amino acid; however, some amino acids are specified by as many as six different codons Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Table 12-3 Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.1 How Is the Information in DNA Used in a Cell? The genetic code uses three bases to specify an amino acid (continued) – Decoding the codons of mRNA is the job of tRNA and ribosomes –Each unique tRNA has three exposed bases, called an anticodon, which are complementary to codon bases in mRNA Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.2 How Is the Information in a Gene Transcribed into RNA? Overview of transcription – Transcription of a DNA gene into RNA has three stages 1. Initiation 2. Elongation 3. Termination Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.2 How Is the Information in a Gene Transcribed into RNA? Overview of transcription (continued) – The three steps of transcription correspond to the three major parts of most genes –A promoter region at the beginning of the gene marks where transcription is to be initiated –The “body” of the gene corresponds with where elongation of the RNA strand occurs –A termination signal at the end of the gene marks where RNA synthesis is to terminate Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.2 How Is the Information in a Gene Transcribed into RNA? Transcription begins when RNA polymerase binds to the promoter of a gene – The enzyme RNA polymerase synthesizes RNA – RNA polymerase binds to the promoter region at the beginning of a gene – The promoter consists of (1) a site that binds RNA polymerase and (2) one or more regulatory sequences that enhance or suppress transcription of the gene – When RNA polymerase binds to the promoter, the DNA molecule is unwound and strands are separated at the beginning of the gene sequence Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Initiation DNA gene 1 gene 2 gene 3 RNA polymerase DNA direction of transcription promoter beginning of gene (3´ end) 1 Initiation: RNA polymerase binds to the promoter region of DNA near the beginning of a gene, separating the double helix near the promoter. Fig. 12-3 (1 of 4) Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.2 How Is the Information in a Gene Transcribed into RNA? Elongation generates a growing strand of RNA – RNA polymerase synthesizes a sequence of RNA nucleotides along one of the DNA strands, the template strand – RNA polymerase travels along the DNA template strand starting at the 3 end of a gene and moving toward the 5 end – The bases in the newly synthesized RNA strand are complementary to the DNA template strand – Starting at the initiation end, the forming RNA strand drifts away from the DNA template strand, while RNA polymerase holds the forming end to the template – As the RNA strand leaves the DNA strands, the helix re-forms Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Elongation RNA DNA template strand 2 Elongation: RNA polymerase travels along the DNA template strand (blue), unwinding the DNA double helix and synthesizing RNA by catalyzing the addition of ribose nucleotides into an RNA molecule (red). The nucleotides in the RNA are complementary to the template strand of the DNA. Fig. 12-3 (2 of 4) Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.2 How Is the Information in a Gene Transcribed into RNA? Transcription stops when RNA polymerase reaches the termination signal – RNA polymerase reaches a termination sequence, releases the completed RNA strand, and detaches from the DNA – The RNA polymerase is then free to bind to the promoter region of another gene and to synthesize another RNA molecule Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Author Animation: Transcription Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Termination and Conclusion of Transcription termination signal 3 Termination: At the end of the gene, RNA polymerase encounters a DNA sequence called a termination signal. RNA polymerase detaches from the DNA and releases the RNA molecule. DNA RNA 4 Conclusion of transcription: After termination, the DNA completely rewinds into a double helix. The RNA molecule is free to move from the nucleus to the cytoplasm for translation, and RNA polymerase may move to another gene and begin transcription once again. Fig. 12-3 (3 & 4 of 4) Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. RNA Transcription in Action gene growing end of RNA gene molecules DNA beginning of gene Fig. 12-4 Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Messenger RNA synthesis differs between prokaryotes and eukaryotes – Messenger RNA synthesis in prokaryotes –All the nucleotides in a gene encode for the amino acids of a protein; there are no sequences that are not transcribed into RNA –Genes for related functions are adjacent and are transcribed together Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Messenger RNA synthesis in prokaryotes (continued) –Because prokaryotes have no nuclear membrane, translation and transcription are not separated in space or time –Ribosomes begin translation at the free 5 end of mRNA, even as RNA polymerase is elongating the mRNA at its 3 end Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Messenger RNA Synthesis in Prokaryotic Cells gene regulating DNA sequences gene 1 gene 2 gene 3 genes coding enzymes in a single metabolic pathway (a) Gene organization on a prokaryotic chromosome DNA mRNA ribosome direction of transcription RNA polymerase DNA mRNA protein ribosome (b) Simultaneous transcription and translation in prokaryotes Biology: Life on Earth, 9e Fig. 12-5 Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Messenger RNA synthesis in eukaryotes – In eukaryotes, the DNA is in the nucleus and the ribosomes are in the cytoplasm – The genes that encode the proteins for a metabolic pathway are not clustered together on the same chromosome Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Messenger RNA synthesis in eukaryotes (continued) – Each gene consists of two or more segments of DNA that encode for protein, called exons, that are interrupted by other segments that are not translated, called introns Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Messenger RNA synthesis in eukaryotes (continued) – Transcription of a gene produces a very long RNA strand that contains introns and exons – This long strand, which extends beyond the first and last exons, is often called precursor mRNA, or premRNA – More nucleotides are added at the beginning and end of the pre-mRNA molecule, forming a “cap” and “tail” – The nucleotides assist with moving the RNA through the nuclear envelope, to bind the mRNA to a ribosome, and to prevent cellular enzymes from breaking down the mRNA Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Messenger RNA synthesis in eukaryotes (continued) – Enzymes in the nucleus cut out the introns and splice together the exons to make true mRNA – The mRNA then exits the nucleus through a membrane pore and associates with a ribosome Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Messenger RNA Synthesis in Eukaryotic Cells exons DNA promoter introns (a) Eukaryotic gene structure DNA 1 Transcription pre-mRNA 2 An RNA cap and tail are added cap tail 3 RNA splicing finished mRNA 4 Finished mRNA is moved to the cytoplasm for translation (b) RNA synthesis and processing in eukaryotes Biology: Life on Earth, 9e introns are cut out and broken down Fig. 12-6 Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Possible functions of intron-exon gene structure – Through alternative splicing of the exons in a gene, a cell can make multiple proteins from a single gene –Alternative splicing represents an exception to Beadle and Tatum’s one gene–one protein relationship Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Possible functions of intron-exon gene structure (continued) – A second function for the presence of intronexon gene structure is that fragmented genes may provide a quick and efficient way for eukaryotes to evolve new proteins with new functions –If breaks in chromosomes occur in introns, exons may remain intact and be spliced to other chromosomes in ways that produce new, useful proteins Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? During translation, mRNA, tRNA, and ribosomes cooperate to synthesize proteins – Like transcription, translation has three steps 1. Initiation 2. Elongation 3. Termination Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Step 1: Initiation 1. A preinitiation complex forms, consisting of the small ribosomal subunit, a methionine tRNA, and several other proteins 2. The UAC anticodon of the methionine tRNA in the preinitiation complex binds the mRNA molecule by base-pairing with the AUG start codon at the beginning (5 end) of the mRNA Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Step 1: Initiation (continued) 3. The large ribosomal subunit attaches to the small subunit, holding the mRNA between the two subunits and holding the methionine tRNA in its first tRNA binding site – The ribosome is fully assembled at this point and ready to begin translation Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Translation Is the Process of Protein Synthesis: Initiation Initiation: amino acid met tRNA preinitiation complex catalytic site met anticodon methionine tRNA U A C small ribosomal subunit second tRNA binding site U A C mRNA GC A U GGU U C A first tRNA binding site large ribosomal subunit U AC GC A UGGU UCA start codon 1 A tRNA with an attached methionine amino acid binds to a small ribosomal subunit, forming a preinitiation complex. 2 The preinitiation complex binds to an mRNA molecule. The methionine (met) tRNA anticodon (UAC) base-pairs with the start codon (AUG) of the mRNA. 3 The large ribosomal subunit binds to the small subunit. The methionine tRNA binds to the first tRNA site on the large subunit. Fig. 12-7 (1-3 of 9) Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Step 2: Elongation 4. A second tRNA anticodon base-pairs with the second codon on the mRNA, as the tRNA is bound to the second tRNA binding site 5. The catalytic site of the large subunit breaks the bond holding methionine to its tRNA and forms a peptide bond between this amino acid and the amino acid attached to the second tRNA Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Step 2: Elongation (continued) 6.The “empty” tRNA is released and the ribosome moves down the mRNA one codon – The tRNA with the growing amino acid chain is now in the ribosome’s first binding site, and the second binding site is empty, awaiting another tRNA Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Step 2: Elongation (continued) 7. A third tRNA, with an anticodon complementary to the third codon of the mRNA, enters the empty second binding site of the ribosome and base-pairs with the mRNA Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Step 2: Elongation (continued) 8. The catalytic site on the large subunit now passes the growing protein chain onto the third amino acid, the empty tRNA leaves the ribosome, and the ribosome shifts to the next codon on the mRNA – This process continues, one codon at a time, until a stop codon is reached Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Step 3: Termination 9. When the ribosome reaches a stop codon in the mRNA molecule, releasing factors cause it to release the completed peptide chain and the mRNA and to disassemble into its large and small subunits – A tRNA does not bind the stop codon Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Author Animation: Translation Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Translation Is the Process of Protein Synthesis: Elongation and Termination Elongation: catalytic site peptide bond U A C C A A U A C C A A G C A U G G U U C A G C A U G G U U C A initiator tRNA detaches C A A G C A U G G U U C A U A G ribosome moves one codon to the right 4 The second codon of mRNA (GUU) base-pairs with the anticodon (CAA) of a second tRNA carrying the amino acid valine (val). This tRNA binds to the second tRNA site on the large subunit. 5 The catalytic site on the large subunit catalyzes the formation of a peptide bond linking the amino acids methionine and valine. The two amino acids are now attached to the tRNA in the second binding site. 6 The "empty" tRNA is released and the ribosome moves down the mRNA, one codon to the right. The tRNA that is attached to the two amino acids is now in the first tRNA binding site and the second tRNA binding site is empty. Fig. 12-7 (4-6 of 9) Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Translation Is the Process of Protein Synthesis: Elongation and Termination Termination: C A A G U A C A A G U A completed peptide stop codon G C A U G G U U C A U A G C A U G G U U C A U A G CGAA UC UAGUAA 7 The third codon of mRNA (CAU) base-pairs with the anticodon (GUA) of a tRNA carrying the amino acid histidine (his). This tRNA enters the second tRNA binding site on the large subunit. 8 The catalytic site forms a peptide bond between valine and histidine, leaving the peptide attached to the tRNA in the second binding site. The tRNA in the first site leaves, and the ribosome moves one codon over on the mRNA. 9 This process repeats until a stop codon is reached; the mRNA and the completed peptide are released from the ribosome, and the subunits separate. Fig. 12-7 (7-9 of 9) Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Summing up – Each DNA gene codes for a single protein – Transcription produces an mRNA strand complementary to the DNA gene template strand Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Summing up (continued) – The mRNA strand leaves the nucleus and associates with a ribosome in the cytoplasm – Transfer RNAs in the cytoplasm are loaded with their appropriate amino acids by cytoplasmic enzymes Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.3 How Is the Base Sequence of Messenger RNA Translated into Protein? Summing up (continued) – The ribosome “selects” the tRNAs on the basis of the base-pairing of the anticodon with the exposed mRNA codon – The mRNA contains a stop codon to define where protein synthesis ends Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Complementary Base-Pairing Is Critical to the Process of Decoding Genetic Information gene (a) DNA A T G G G A G T T complementary DNA strand template DNA strand etc. T A C C C T C A A etc. codons A U G G G A G U U etc. (b) mRNA anticodons (c) tRNA U A C C C U C A A etc. amino acids (d) protein Biology: Life on Earth, 9e methionine glycine valine etc. Fig. 12-8 Copyright © 2011 Pearson Education Inc. 12.4 How Do Mutations Affect Protein Function? Mutations are changes in the base sequence of DNA caused by mistakes during replication or by various environmental factors Mutations take many forms and can affect protein function in many ways – Mutations fall into five categories – Inversions – Translocations – Deletions – Insertions – Substitutions Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.4 How Do Mutations Affect Protein Function? Mutations take many forms and can affect protein function in many ways (continued) – Inversions and translocations occur when pieces of DNA are broken apart and reattached, within a single chromosome or to a different chromosome – These mutations may be relatively benign if entire genes are merely moved from one place to another – However, if a gene is split in two, it will no longer code for a complete, functional protein – Severe hemophilia is often caused by an inversion in the gene that encodes a protein required for blood clotting Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.4 How Do Mutations Affect Protein Function? Mutations take many forms and can affect protein function in many ways (continued) – A deletion occurs when one or more nucleotides are removed from the gene sequence – An insertion occurs when one or more nucleotides are added to the gene sequence Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.4 How Do Mutations Affect Protein Function? Deletions and insertions (continued) – Depending on how many nucleotides are involved, deletions and insertions can cause a misreading of a gene’s codons during transcription or replication –The codons in THEDOGSAWTHECAT is changed by deletion of the letter “E” to THD OGS AWT HEC AT –Such mutations are called frameshift mutations Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.4 How Do Mutations Affect Protein Function? Deletions and insertions (continued) – Proteins that result from deletions and insertions have a very different amino acid sequence and almost always are nonfunctional – Deletions and insertions of three nucleotides (or a multiple of three) do not cause a shift of the reading frame and, so, may simply subtract or add a harmless amino acid to the protein – The defective myostatin gene of Belgian Blue cattle has an 11-nucleotide deletion, generating a premature stop codon Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.4 How Do Mutations Affect Protein Function? Mutations take many forms and can affect protein function in many ways (continued) – When a nucleotide substitution (point mutation) occurs, an incorrect nucleotide takes the place of a correct one – A point mutation sometimes does not change the amino acid sequence of the protein – Because many amino acids are encoded by more than one codon, the mutation may cause the same amino acid to be added – A known point mutation in the beta-globin gene for hemoglobin causes CTC to change to CTT, but since both codons code for glutamic acid, the protein is unchanged Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.4 How Do Mutations Affect Protein Function? With some nucleotide substitutions, the substituted amino acid may be functionally equivalent to the normal one, allowing the mutated protein to function normally – In beta-globin, the amino acids on the outside of the protein must be hydrophilic to keep the protein dissolved in the cytoplasm of red blood cells, but which hydrophilic amino acids are on the outside doesn’t matter much – In beta-globin, a point mutation of the CTC codon to GTC causes glutamic acid (hydrophilic) to be replaced with glutamine (also hydrophilic), but the resulting protein functions well – Substitutions causing no amino acid changes or changes that are unimportant to function are called neutral mutations Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.4 How Do Mutations Affect Protein Function? Some substitutions cause an altered amino acid sequence that change protein function dramatically, usually for the worse –The substitution of an adenine for a thymine in the CTC CAC mutation in a hemoglobin gene causes valine (hydrophobic) to replace glutamic acid (hydrophilic) –Placing this hydrophobic amino acid on the outside of the hemoglobin molecule leads to the clumping of hemoglobin and distortion of the red blood cell seen in sickle cell anemia Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.4 How Do Mutations Affect Protein Function? When a nucleotide substitution (point mutation) occurs, an incorrect nucleotide takes the place of a correct one (continued) – The point mutation may introduce a premature stop codon, leading to an mRNA that produces an incomplete protein – Such a mutation in the beta-globin gene prevents production of functional beta-globin protein – This leads to beta-thalassemia – People with this mutation have only alpha-globin subunits and require frequent blood transfusions to survive Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Table 12-4 Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.4 How Do Mutations Affect Protein Function? Mutations provide the raw material for evolution – Mutations are heritable changes in the DNA – Approx. 1 in 105–106 eggs or sperm carry a mutation – Most mutations are harmful or neutral Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.4 How Do Mutations Affect Protein Function? Mutations provide the raw material for evolution (continued) – Mutations create new gene sequences and are the ultimate source of genetic variation – Mutant gene sequences that are beneficial may spread through a population and become common Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? The human genome contains 20,000 to 25,000 genes A given cell “expresses” (transcribes) only a small number of genes Some genes are expressed in all cells, such as genes coding for tRNAs, since all cells require proteins Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? Other genes are expressed only in certain types of cells, at certain times in an organism’s life, or under specific environmental conditions –For example, even though every cell in your body contains the gene for casein, the major protein in milk, this gene is expressed only in certain cells in the breast, only in mature women, and only when a woman is breastfeeding Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? Regulation of gene expression may occur at three different levels 1. At the level of transcription, regulation determines which genes in a cell are expressed 2. At the level of translation, regulation determines how much protein is made from a particular type of mRNA 3. At the level of protein activity, regulation determines how long the protein lasts in a cell and how rapidly protein enzymes catalyze specific reactions Although these general principles apply to both prokaryotic and eukaryotic organisms, there are some differences as well Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? Gene regulation in prokaryotes – Prokaryotic DNA is organized into units called operons, which contain functionally related genes Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? Gene regulation in prokaryotes (continued) – Each operon consists of four parts – A regulatory gene controls the timing and rate of transcription of the other genes – A promoter is the site that RNA polymerase recognizes as the place to start transcription – An operator governs the access of RNA polymerase to the promoter – The structural genes actually encode the related enzymes or other proteins Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? Gene regulation in prokaryotes (continued) – Whole operons are regulated as units, so that functionally related proteins are synthesized simultaneously when the need arises Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? Gene regulation in prokaryotes (continued) – The intestinal bacterium Escherichia coli (E. coli) lives on what its host eats – Specific enzymes are needed to metabolize the type of food that comes along – For example, in newborn mammals, E. coli are bathed in milk, which contains the milk sugar lactose – The lactose operon contains three structural genes, each coding for an enzyme that aids in lactose metabolism Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? Gene regulation in prokaryotes (continued) – In the absence of lactose, the lactose operon is normally shut off, or repressed, by a repressor protein – The regulatory gene of the lactose operon directs the synthesis of this protein, which represses the operon by binding to the operator site – Under these circumstances, RNA polymerase, although able to find to the promoter, cannot get past the repressor protein to transcribe the structural genes – Consequently, lactose-metabolizing enzymes are not synthesized Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? Gene regulation in prokaryotes (continued) – When lactose is present, it binds the repressor and changes the repressor’s shape – When bound to lactose, the repressor’s altered shape does not permit it to bind the operator – Without the repressor in the operator, RNA polymerase is able to reach the promoter and begin transcription of the genes needed to metabolize lactose – When lactose is no longer present, the repressor resumes its inhibitory conformation and binds the operator, thus blocking transcription again Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Regulation of the Lactose Operon regulatory gene: codes for repressor protein R P operator: repressor protein binds here O gene 1 gene 2 gene 3 structural genes that code for promoter: RNA enzymes of lactose metabolism polymerase binds here (a) Structure of the lactose operon RNA polymerase transcription blocked R P gene 1 gene 2 gene 3 a repressor protein bound to the operator site overlaps the promoter free repressor proteins (b) Lactose absent RNA polymerase binds to the promoter and transcribes the structural genes R O gene 1 lactose bound to repressor proteins (c) Lactose present Biology: Life on Earth, 9e gene 2 gene 3 lactose-metabolizing enzymes are synthesized Fig. 12-9 Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? Gene regulation in eukaryotes – Although eukaryotic gene regulation bears some similarity to regulation in prokaryotes, the complexity of eukaryotic cells leads to differences as well – DNA is in a membrane-bound nucleus – There are a variety of cell types in multicellular eukaryotes – The genome of eukaryotes is organized differently – RNA transcripts undergo complex processing not seen in prokaryotes Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? Gene regulation in eukaryotes (continued) – Expression of genetic information by a eukaryotic cell is a multistep process, beginning with transcription of DNA and ending with a protein that performs a particular function – Regulation can occur at any of these steps Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? Gene expression in eukaryotes can be regulated at a number of points 1.Cells can control the frequency at which an individual gene is transcribed 2.The same gene may be used to produce different mRNAs and protein products – Differential splicing of exons yields different proteins, according to a cell’s needs Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? Gene expression in eukaryotes can be regulated at a number of points (continued) 3.Cells may control the stability and translation of messenger RNAs – Some mRNAs are long-lasting and translated into protein many times; others are translated only a few times before being degraded – Small “regulatory RNA” molecules may block translation of some mRNAs, or may even target some mRNAs for destruction Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? Gene expression in eukaryotes can be regulated at a number of points (continued) 4. Proteins may require modification before they can carry out their functions – Some modifications of the inactive enzyme involve excising a segment of it, thus exposing the active site and activating the enzyme – Another common method of changing an enzyme’s activity is phosphorylating or dephosphorylating it – Controlling these modifications provides a way to regulate the enzymes Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? Gene expression in eukaryotes can be regulated at a number of points (continued) 5.Cells can control the rate at which proteins are degraded – By preventing or promoting a protein’s degradation, a cell can rapidly adjust the amount of a particular protein it contains Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. An Overview of Information Flow in a Eukaryotic Cell DNA 1 Transcription rRNA proteins (nucleus) pre-mRNA Cells can control the frequency of transcription tRNA 2 mRNA processing Different mRNAs may be produced from a single gene mRNA (cytoplasm) ribosomes mRNA tRNA amino acids 3 Translation If the active protein is an enzyme, it will catalyze a chemical reaction in the cell inactive protein Cells can control the stability and rate of translation of particular mRNAs Modification Cells can regulate a protein’s activity by modifying it 5 Degradation Cells can regulate a protein’s activity by degrading it 4 substrate active protein product amino acids Fig. 12-10 Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? In eukaryotic cells, transcriptional regulation occurs on at least three levels – The individual gene – Regions of chromosomes – Entire chromosomes Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? In eukaryotic cells, transcriptional regulation occurs on at least three levels (continued) – Because most gene promoters contain transcription factor binding sites, or response elements, transcription can be regulated by the presence of transcription factors –Free radicals cause the production of a transcription factor that binds a free radical response element in the promoter of a gene that degrades free radicals Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? Because most gene promoters contain transcription factor binding sites, or response elements, transcription can be regulated by the activity of transcription factors (continued) – When egg production needs to be increased in birds, the ovaries release estrogen – The estrogen forms a complex with (and thus activates) a transcription factor called estrogen receptor – The complex then binds the estrogen response element in the albumin gene, making it easier for RNA polymerase to bind to the promoter and initiate transcription of mRNA – The mRNA is then translated into large amounts of albumin Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? In eukaryotic cells, transcriptional regulation occurs on at least three levels (continued) – Condensed or tightly wound regions of DNA can make genes inaccessible to RNA polymerase –Some condensed portions of chromosomes contain structural elements of the chromosome, but no genes –Other condensed areas contain genes that the cell may not currently need and will decondense when the genes are needed Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? In eukaryotic cells, transcriptional regulation occurs on at least three levels (continued) – Large parts of chromosomes may be inactivated, preventing transcription – In female mammals, one entire X chromosome is condensed – In 1961, the geneticist Mary Lyon hypothesized that one of the two X chromosomes in females was inactivated in some way, so that its genes were not expressed – These so-called Barr bodies were discovered by Murray Barr Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. A Barr Body Fig. 12-11 Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? In female mammals, one entire X chromosome is condensed (continued) – We now know that about 85% of the genes on an inactivated X chromosome are not transcribed – Early in development (about the 16th day in humans), one X chromosome in each of a female’s cells begins to produce large amounts of a regulatory RNA molecule called Xist –Xist coats most of the chromosome, condenses it into a tight mass, and prevents transcription Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? In female mammals, one entire X chromosome is condensed (continued) – Because Barr bodies are formed early in development, female mammals have large clusters of cells with one X chromosome inactivated and other clusters of cells in which the other X chromosome is inactivated –Each cluster is descended from a single cell of the very early embryo Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. 12.5 How Are Genes Regulated? In female mammals, one entire X chromosome is condensed (continued) – This effect can be observed in the fur patterns of calico cats – The X chromosome of a cat contains a gene for fur pigmentation – Different patches of skin cells in a cat inactivate different X chromosomes – If there are two alleles for fur color in the cat’s genotype, the cat will produce patches of different fur color (usually orange and black), depending on which X chromosome was condensed in the progenitor cell that gave rise to each patch of cells Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc. Inactivation of the X Chromosome Regulates Gene Expression Fig. 12-12 Biology: Life on Earth, 9e Copyright © 2011 Pearson Education Inc.