Transcription & Translation. From Gene to Protein

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1 Transcription & Translation From Gene to Protein

2 Part 1 A little history lesson

3 In 1909, 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 Linking genes to enzymes required understanding that cells synthesize and degrade molecules in a series of steps, a metabolic pathway

4 Nutritional Mutants in Neurospora: Scientific Inquiry George Beadle and Edward Tatum exposed bread mold to X-rays, creating mutants that were unable to survive on minimal medium as a result of inability to synthesize certain molecules Using crosses, they 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

5 However, some proteins aren t enzymes, so researchers later revised the hypothesis: one gene one protein Many proteins are composed of several polypeptides, each of which has its own gene Therefore, Beadle and Tatum s hypothesis is now restated as the one gene one polypeptide hypothesis Note that it is common to refer to gene products as proteins rather than polypeptides

6 Part 2 A Basic Overview

7 Background The process that describes how enzymes and other proteins are made from DNA is called protein synthesis. Protein Synthesis has 3 steps: Transcription mrna is created from a strand of DNA RNA processing mrna is edited Translation mrna is read by a ribosome and used to assemble amino acids into polypeptides

8 Types of RNA mrna Messenger RNA A single strand of RNA that provides a template used for sequencing amino acids into a polypeptide. A triplet of 3 adjacent nucleotides on the mrna, called a codon, codes for one specific amino acid. Since there are 64 possible ways that 4 nucleotides can be arranged in triplet combinations, there are 64 possible codons. However, there are only 20 amino acids. Thus, some codons code for the same amino acid.

9 Genetic Code A visual representation of the possible codon combinations and the amino acids each codon codes for.

10 First mrna base (5 end of codon) Third mrna base (3 end of codon) Fig Second mrna base

11 Genetic Code

12 Pinwheel Genetic Code

13 Types of RNA trna Transfer RNA A short RNA molecule used for transporting amino acids to their proper place on the mrna template. There are about 45 different trnas Due to interactions between various parts of the trna molecule, it is folded and looks like the 3 leaflets of a clover leaf. Contains the anticodon that allows it to bind to mrna. Example: if mrna has the codon AUG, trna will have the complimentary anticodon UAC. This codon-anticodon arrangement allows the trna to connect to the mrna.

14 Types of RNA rrna ribosomal RNA The building blocks of ribosomes In the nucleolus, various proteins are imported from the cytoplasm and assembled with rrna to form large and small ribosomal subunits. Together these two subunits form a ribosome that coordinates the activities of mrna and trna during translation.

15 Transcription

16 3 Parts 1.) Initiation RNA polymerase attaches to a promoter region on the DNA and begins to unzip the two strands. The promotor region for mrna transcriptions often contains the sequence T-A-T-A. called the TATA Box.

17 2.) Elongation Occurs as the RNA pol unzips the DNA and assembles RNA nucleotides using one side of the DNA as a template. Elongation occurs in the 5 3 direction

18 3.) Termination Occurs when the RNA pol reaches a special sequence of nucleotides that serve as a termination point. In eukaryotes, the termination region often contains the DNA sequence AAAAAAA.

19 Fig. 17-7a-1 Promoter Transcription unit 5 3 Start point RNA polymerase DNA 3 5

20 Fig. 17-7a-2 Promoter Transcription unit 5 3 Start point RNA polymerase DNA 1 Initiation Unwound DNA RNA transcript Template strand of DNA 3 5

21 Fig. 17-7a-3 Promoter Transcription unit 5 3 Start point RNA polymerase DNA 1 Initiation Unwound DNA RNA transcript Template strand of DNA Elongation Rewound DNA RNA transcript

22 Fig. 17-7a-4 Promoter Transcription unit 5 3 Start point RNA polymerase DNA 1 Initiation Unwound DNA RNA transcript Template strand of DNA Elongation Rewound DNA RNA transcript 3 Termination Completed RNA transcript 3 3 5

23 Fig. 17-7b Elongation Nontemplate strand of DNA RNA polymerase RNA nucleotides 3 3 end 5 5 Newly made RNA Direction of transcription ( downstream ) Template strand of DNA

24 Animation Transcription Animation

25 mrna Processing Editing the message

26 Steps of processing 1.) The 5 cap (-P-P-P-G-5 )_ A cap is added to the 5 end of the mrna. The cap is a guanine nucleotide with 2 additional phosphate groups, forming GTP (similar to ATP). This capping gives stability to the mrna and an attachment point for the ribosome. 2.) The poly-a tail (-A-A-A.A-A-3 ) A poly-a tail is added to the 3 end of the mrna. The tail consists of about 200 adenine nucleotides. Provides stability and aids in the mrna passing through the nuclear envelope.

27 3.) RNA Splicing Nucleotide segments are removed from the mrna DNA segments contain both coding and noncoding sequences. The coding segments = exons; The non-coding segments = introns The original unprocessed mrna contains both the coding and non-coding sequences. The introns have to be cut out and the exons have to be spliced together in order to create an mrna with a continuous coding sequence.

28 Fig Pre-mRNA 5 Cap 5 Exon Intron Exon Intron 105 Exon Poly-A tail Coding segment Introns cut out and exons spliced together mrna 5 Cap UTR 3 UTR Poly-A tail

29 In some cases, RNA splicing is carried out by spliceosomes Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snrnps) that recognize the splice sites

30 Fig RNA transcript (pre-mrna) Exon 1 Intron Exon 2 Protein snrna snrnps Other proteins

31 Fig RNA transcript (pre-mrna) Exon 1 Intron Exon 2 Protein snrna snrnps Other proteins Spliceosome 5

32 Fig RNA transcript (pre-mrna) Exon 1 Intron Exon 2 Protein snrna snrnps Spliceosome Other proteins 5 Spliceosome components 5 mrna Exon 1 Exon 2 Cut-out intron

33 Animation RNA Splicing Animation Splicing Animation #2 Another RNA Splicing Animation

34 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

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

36 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 RNA splicing Such variations are called alternative RNA splicing Because of alternative splicing, the number of different proteins an organism can produce is much greater than its number of genes

37 Translation Making a protein

38 Background After transcription, the mrna, trna and ribosomal subunits are transported across the nuclear envelope and into the cytoplasm. In the cytoplasm, the amino acids attach to the 3 end of the trnas, forming an aminoacyl-trna.

39 Background The reaction to attach the amino acid to the trna requires an enzyme specific to each trna and the energy from one ATP.

40 As in transcription, translation is categorized into 3 steps: Initiation Elongation Termination The energy for translation is provided by several GTP molecules. GTP acts as an energy supplier in the same manner as ATP

41 A ribosome has three binding sites for trna: The P site holds the trna that carries the growing polypeptide chain The A site holds the trna that carries the next amino acid to be added to the chain The E site is the exit site, where discharged trnas leave the ribosome Copyright 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

42 Steps of translation 1.) Initiation begins when the small ribosomal subunit attaches to a special region near the 5 end of the mrna. 2.) A trna (with the anticodon UAC) carrying the amino acid methionine attaches to the mrna at the start codon, AUG, on a spot on the ribosome called the P site

43 Fig U 5 A A U C5 G3 P site Large ribosomal subunit Initiator trna 5 mrna Start codon mrna binding site 3 GTP Small ribosomal subunit GDP E A 5 3 Translation initiation complex

44 3.) Another trna carrying another amino acid comes in and binds to the mrna at the A site. 4.) The amino acid from the trna in the P site is moved to the amino acid on the trna in the A site. This is called Elongation

45 5.) The mrna moves over one position. The first trna now occupies the E site, the second trna (with the growing amino acid chain) now ocupies the P site and the A site is open for the next trna. 6.) The first trna is ejected from the E site and goes into the cytoplasm to get another amino acid.

46 Fig b P site (Peptidyl-tRNA binding site) E site (Exit site) mrna binding site E P A site (AminoacyltRNA binding site) Large subunit Small subunit (b) Schematic model showing binding sites A Amino end mrna 5 E Codons Growing polypeptide Next amino acid to be added to polypeptide chain trna 3 (c) Schematic model with mrna and trna

47 Fig Amino end of polypeptide mrna 5 E P site A site 3

48 Fig Amino end of polypeptide mrna 5 E P site A site 3 GTP GDP E P A

49 Fig Amino end of polypeptide mrna 5 E P site A site 3 GTP GDP E P A E P A

50 Fig Amino end of polypeptide mrna E 3 Ribosome ready for next aminoacyl trna 5 P site A site GTP GDP E E P A P A GDP GTP E P A

51 Termination of Translation Termination occurs when a stop codon in the mrna reaches the A site of the ribosome. The A site accepts a protein called a release factor. The release factor causes the addition of a water molecule instead of an amino acid. This reaction releases the polypeptide, and the translation assembly then comes apart. Copyright 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings

52 Fig Release factor 3 5 Stop codon (UAG, UAA, or UGA)

53 Fig Release factor Free polypeptide Stop codon (UAG, UAA, or UGA) 3 2 GTP 2 GDP

54 Fig Release factor Free polypeptide Stop codon (UAG, UAA, or UGA) 3 2 GTP 5 2 GDP 3

55 Animation Translation Animation

56 Mutations revisited

57 Types of mutations Point mutation: Examples: insertion, deletion, substitution, frameshift (results from insertion or deletion)

58 Effects of mutations 1.) Silent mutation Has no effect, because the new codon codes for the same amino acid as the old codon. Example: CUU, CUG, CUA, CUC all code for the amino acid Leucine. So long as the 3 rd nucleotide is the only one that is changed, the effect is zero.

59 2.) Missense Mutation The mutation causes a new codon that codes for a new amino acid. This may have only a minor effect or it may result in the production of a protein that is unable to form into its proper 3-D shape and, therefore, is unable to carry out its normal function. The hemoglobin protein that causes sickle-cell disease is caused by a missense mutation.

60 3.) Non-sense Mutation Occurs when the new codon is a stop codon.

61 DNA Organization

62 Background In eukaryotes, DNA is packaged with proteins to form a matrix called chromatin The DNA is coiled around bundles of 8-9 histone proteins to form DNA-histone complexes called nucleosomes. Microscopically, nucleosomes look like beads on a string.

63 Fig a DNA double helix (2 nm in diameter) Histones Nucleosome (10 nm in diameter) Histone tail H1 DNA, the double helix Histones Nucleosomes, or beads on a string (10-nm fiber)

64 Fig b Chromatid (700 nm) 30-nm fiber Loops Scaffold 300-nm fiber Replicated chromosome (1,400 nm) 30-nm fiber Looped domains (300-nm fiber) Metaphase chromosome

65 During cell division, DNA is compactly organized into chromosomes. When the cell is not dividing, there are 2 types of chromatin: 1.) Euchromatin DNA is loosely bound to histones. DNA here is actively being transcribed. 2.) Heterochromatin DNA is tightly bound to the histones. DNA is inactive in these regions.

66 What in the wide, wide world of sports is a goin on here?!!? Jumping genes?

67 Yep Some DNA segments within genes are able to move to new locations. This isn t really a good thing. These transposible genetic elements, called transposons (or jumping genes) can move to a new location on the same chromosome or to a different chromosome.

68 History Discovered in the 1940s by Barbara McClintock. She also discovered that crossing over in meiosis was a thing. A super important scientist most people have NEVER heard of. She was studying maize. Findings: Parts of chromosomes can move randomly to other locations on the chromosome, affecting phenotypic expression. Genes can be turned on/off by environmental factors. Genetic disorders can be reversed (in maize).

69 Some transposons consist only of DNA that codes for an enzyme that enables it to be transported. Other transposons contain genes that invoke the replication of the transposon. After replication, the new transposon copy is transported to the new location. Wherever they are inserted, transposons have the effect of a mutation. They may change the expression of a gene, turn on or off its expression, or have no effect at all.

70 Stats: McClintock found that approx. 90% of maize DNA consists of transposons. 44% in humans

71 In humans Most common transposon is the Alu sequence. 300 bp long. Occurs over 1 million different times in human genome. Very common sequence (approx. 17% of total genome). Insertions in Alu sequence in humans generally have no effect b/c most of the sequences occur in introns.

72 In humans ACE gene (Angiotensin Converting Enzyme) Comes in 2 varieties, one WITH an Alu insertion, and one WITHOUT. Variation is linked to sporting performance: With = better at endurance events (distance running, biking, distance swimming, etc). Without = better at strength/power events (weightlifting, wrestling, etc).

73 In humans Opsin gene duplication in Old World Primates (including humans) is hypothesized for the regaining of trichromacy (3-color vision). Birds/fish have 4-color vision. Most other mammals are dichromatic.

74 The Molecular Genetics of Viruses

75 Background Viruses are parasites of cells. Typical mode of infection: 1.) A typical virus penetrates a cell, 2.) it takes over the cell s metabolic machinery, 3.) The virus (using the cell s machinery) assembles hundreds of new viruses that are copies of itself. 4.) Viruses then leave the cell (usually by destroying the host cell) and infect other cells.

76 Viruses are specific for the kinds of cells they will parasitize. Some viruses only attack one type of cell within a single host species. Others attack similar cells from a range of closely related species. Bacteriophages, or phages, are viruses that attack only bacteria.

77 Viral Structure Viruses consist of the following structures: A Nucleic Acid Either DNA or RNA (not both) Contains the hereditary info of the virus May be double stranded (dsdna or dsrna) or single stranded (ssdna or ssrna) A Capsid A protein coat that encloses the nucleic acid. Identical protein subunits, called capsomeres, assemble to form the capsid.

78 Viral Structure Some viruses have an envelope that surrounds the capsid. The envelopes incorporate phospholipids and proteins obtained from the cell membrane of the host. Why would this be advantageous to the virus?

79 Viral Structure Bacteriophage Structure Animal Virus Structure Enveloped Virus Structure

80 Types of Viruses

81 Viral Replication Two ways: 1.) Lytic Cycle The virus penetrates the cell membrane of the host cell and takes over the host cell. Once the viral particles have been replicated, the host cell ruptures, releasing the viral particles.

82 2.) Lysogenic Cycle viral DNA is temporarily incorporated into the host cell DNA. A virus in this dormant stage is called a provirus or if a bacteriophage a prophage. As the cell goes through mitosis, it copies the virus as well. The virus remains inactive until some environmental trigger causes the virus to begin the destructive lytic cycle. Triggers = radiation, chemicals.

83

84 Retroviruses ssrna viruses that use an enzyme called reverse transcriptase to make a DNA complement of their RNA. The DNA complement can then be transcribed immediately to manufacture mrna (to make new viral proteins) or it can begin the lysogenic cycle. HIV works this way. Life cycle of HIV

85 The Molecular Genetics of Bacteria

86 Background Bacteria have Cell walls Cell membranes Ribosomes DNA In a single circular chromosome Bacteria lack Nucleus Specialized organelles Histones

87 Bacterial Chromosome Is often called a naked chromosome because it lacks the histones and other proteins associated with eukaryotic chromosomes.

88 Bacterial Plasmids Bacteria contain short, circular DNA molecules outside the chromosome called plasmids. Plasmids carry genes that are beneficial but not normally vital for survival of the organism. Plasmids replicate independently of the chromosome. Some plasmids, called episomes, can become incorporated into the bacterial chromosome.

89 Genetic Variation in Bacteria Bacteria can alter their genome in 3 ways: 1.) Conjugation DNA exchange between bacteria. A donor bacterium produces a tube, called a Sex Pilus, that connects to another bacterium. The donor sends chromosomal or plasmid DNA to the recipient through the pilus. In some cases, large portions of a donor s chromosome are sent, thus allowing recombination with the recipient s chromosome.

90 Genetic Variation in Bacteria Two plasmids in conjugation: 1.) F Plasmid Contains the genes needed to produce pili (plural for pilus). When the recipient bacterium receives the F plasmid, it too can become a donor cell. 2.) R plasmid Provide bacteria with antibiotic resistance.

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93 Genetic Variation in Bacteria Bacteria can alter their genome in 3 ways: 2.) Transduction Occurs when new DNA enters the bacterial genome by way of a virus (bacteriophage). When a virus is assembled during the lytic cycle, it is sometimes assembled with some bacterial DNA in place of some viral DNA. When the new virus particles infect another cell, the bacterial DNA they carry can recombine with the resident DNA.

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95 Genetic Variation in Bacteria Bacteria can alter their genome in 3 ways: 3.) Transformation Occurs when bacteria absorb DNA from their surroundings and incorporate it into their genome. Some bacteria have specialized proteins on their cell membranes that allow this to occur.

96

97 Just for fun

98 Regulation of Gene Expression

99 Background Every cell in a human contains exactly the same sequences of DNA. Yet, some cells become muscle cells and other cells become nerve cells. One way that cells with identical DNA become different is by regulating gene expression through transcription of only selected genes. This is called epigenetics

100 Prokaryotic Gene Expression In prokaryotes, an operon is a unit of DNA that controls gene transcription. Operons contain the following parts: 1.) Promoter 2.) Operator 3.) Structural Gene 4.) Regulatory Gene

101 Promoter A sequence of DNA to which the RNA polymerase attaches to begin transcription. Operator a region of DNA that can block the action of RNA polymerase if the region is occupied by a repressor protein.

102 Structural Gene Contain DNA sequences that code for several related enzymes that direct the production of a particular end product. Regulatory Gene Is outside the operon region Produces repressor proteins and activator proteins

103 Repressor Protein Substances that occupy the operator region and block the action of RNA polymerase. Activator Protein Assist the attachment of RNA polymerase to the promotor region

104 Examples of Operons 1.) lac operon In E. coli Controls the breakdown of lactose. Enzymes produced by the operon are inducible enzymes. 2.) trp operon In E. coli Controls the production of the amino acid tryptophan Enzymes produced by the operon are repressible enzymes.

105 Video 1 Operons

106 Eukaryotic Gene Expression 3 methods: 1.) Regulatory Proteins Operate similarly to those in prokaryotes. Influence how readily the RNA pol will attach to the promoter region.

107 2.) Nucleosome Packing Influences whether a section of DNA will be transcribed. DNA segments are tightly packed by methylation of the histones, which often makes the region untranscribable. Barr Bodies are highly methylated DNA segments are loosely packed by acetylation of the histones, which allows the DNA to be transcribed. Heterochromatin & Euchromatin

108 3.) RNA Interference Occurs when short interfering RNAs (sirnas) block mrna transcription or translation. Under certain circumstances, an RNA molecule will fold back and base pair with itself, forming dsrna. An enzyme then cuts the dsrna into short pieces, which then base pair to complimentary DNA regions, preventing further transcription of the gene. Sometimes the sirnas will bind to existing mrna strands, thereby preventing ribosomes from binding to the mrna. This effectively inactivates the mrna.

109 4.) Alternative RNA Splicing Certain codons are copied from a gene when other codons in same gene are not. This allows the same gene to code for many different proteins. We ve discussed this before.

110 5.) Protein degradation As proteins age, they lose functionality due to bond interferences. Proteins that need to be recycled due to nonfunction are marked for destruction with a protein called ubiquitin (so called because it is ubiquitous, found in all eukaryotic cells).

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