III. DNA, Protein Synthesis, and Mitosis A. DNA and RNA Structure DNA is the genetic material in all forms of life (eubacteria, archaea, protists, plants, fungi, and animals). Those quasi-living viruses vary in their genetic material. Some have double-stranded DNA (ds-DNA) like living systems, while others have ss-DNA, ss-RNA, and ds-RNA. RNA performs a wide array of functions in living systems. Many of these functions have only been discovered in the last few years.

A. DNA and RNA Structure 1. monomers are “nucleotides” three parts: - pentose sugar (ribose in RNA, deoxyribose in DNA)

A. DNA and RNA Structure 1. monomers are “nucleotides” three parts: - pentose sugar (ribose in RNA, deoxyribose in DNA) - nitrogenous base (A, C, G, U in RNA A, C, G, T in DNA)

A. DNA and RNA Structure 1. monomers are “nucleotides” three parts: - pentose sugar - nitrogenous base Nitrogenous base binds to the 1’ carbon

A. DNA and RNA Structure 1. monomers are “nucleotides” three parts: - pentose sugar - nitrogenous base - phosphate group PO4 binds to the 5’ carbon

A. DNA and RNA Structure 1. monomers are “nucleotides” 2. polymerization occurs by ‘dehydration synthesis’ Between the PO4 (which always has free H+ ions binding and unbinding) of the free nucleotide and the –OH group on the 3’ carbon of the last sugar in the chain. OH OH O-P-O O H2O OH Energy released by cleaving the diphosphate group can be used to power the dehydration synthesis reaction

A. DNA and RNA Structure 1. monomers are “nucleotides” 2. polymerization occurs by ‘dehydration synthesis’ Polymerization results in a polymer of DNA (or RNA). This single polymer is a single-stranded helix It has a ‘polarity’ or ‘directionality’; it has different ends… there is a reactive phosphate at one end (5’) and a reactive –OH at the other (3’). So, the helix has a 5’-3’ polarity. 5’ 3’

A. DNA and RNA Structure 1. monomers are “nucleotides” 2. polymerization occurs by ‘dehydration synthesis’ 3. most DNA exists as a ‘double-helix’ (ds-DNA) (although some viruses have genetic material that is signle-stranded DNA (ss-DNA)) a. The nitrogenous bases on the two helices are ‘complementary’ to one another, and form weak hydrogen bonds between the helices. A purine (A or G) always binds with a pyrimidine (T or C) In fact, A with T (2 h-bonds) And G with C (3 h-bonds)

A. DNA and RNA Structure 1. monomers are “nucleotides” 2. polymerization occurs by ‘dehydration synthesis’ 3. most DNA exists as a ‘double-helix’ (ds-DNA) a. bases are complementary b. the strands are anti-parallel: they are aligned with opposite polarity 5’

A. DNA and RNA Structure 1. monomers are “nucleotides” 2. polymerization occurs by ‘dehydration synthesis’ 3. most DNA exists as a ‘double-helix’ (ds-DNA) 4. RNA performs a wide variety of functions in living cells: a. m-RNA is a ‘copy’ of a gene, read by the ribosome to make a protein

A. DNA and RNA Structure 1. monomers are “nucleotides” 2. polymerization occurs by ‘dehydration synthesis’ 3. most DNA exists as a ‘double-helix’ (ds-DNA) 4. RNA performs a wide variety of functions in living cells: a. m-RNA is a ‘copy’ of a gene, read by the ribosome to make a protein b. R-RNA is made the same way, is IN the Ribosome, and ‘reads’ the m-RNA

A. DNA and RNA Structure 1. monomers are “nucleotides” 2. polymerization occurs by ‘dehydration synthesis’ 3. most DNA exists as a ‘double-helix’ (ds-DNA) 4. RNA performs a wide variety of functions in living cells: a. m-RNA is a ‘copy’ of a gene, read by the ribosome to make a protein b. r-RNA is made the same way, is IN the Ribosome, and ‘reads’ the m-RNA c. t-RNA is made the same way, and brings amino acids to the ribosome

A. DNA and RNA Structure 1. monomers are “nucleotides” 2. polymerization occurs by ‘dehydration synthesis’ 3. most DNA exists as a ‘double-helix’ (ds-DNA) 4. RNA performs a wide variety of functions in living cells: a. m-RNA is a ‘copy’ of a gene, read by the ribosome to make a protein b. r-RNA is made the same way, is IN the Ribosome, and ‘reads’ the m-RNA c. t-RNA is made the same way, and brings amino acids to the ribosome d. mi-RNA (micro-RNA) and si-RNA (small interfering RNA) bind to m-RNA and splice it; inhibiting the synthesis of its protein. This is a regulatory function.

Cell Biology B. Protein Synthesis

B. Protein Synthesis Why is this important? Well…what do proteins DO?

Protein Synthesis A. Overview A T G C T G A C T A C T G T A C G A CT G A T G A C Genes are read by enzymes and RNA molecules are produced… this is TRANSCRIPTION (t-RNA) (r-RNA) U G C U G A C U A C U (m-RNA)

Protein Synthesis A. Overview A T G C T G A C T A C T G T A C G A CT G A T G A C Genes are read by enzymes and RNA molecules are produced… this is TRANSCRIPTION (t-RNA) (r-RNA) U G C U G A C U A C U (m-RNA) Eukaryotic RNA and some prokaryotic RNA have regions cut out… this is RNA SPLICING

R-RNA is complexed with proteins to form ribosomes. Protein Synthesis A. Overview A T G C T G A C T A C T G T A C G A CT G A T G A C R-RNA is complexed with proteins to form ribosomes. Specific t-RNA’s bind to specific amino acids. (t-RNA) (r-RNA) U G C U G A C U A C U Amino acid (m-RNA) ribosome

Protein Synthesis A. Overview A T G C T G A C T A C T G T A C G A CT G A T G A C The ribosome reads the m-RNA. Based on the sequence of nitrogenous bases in the m-RNA, a specific sequence of amino acids (carried to the ribosome by t-RNA’s) is linked together to form a protein. This is TRANSLATION. (t-RNA) (r-RNA) U G C U G A C U A C U Amino acid (m-RNA) ribosome

POST-TRANSLATIONAL MODIFICATION. (t-RNA) (r-RNA) Protein Synthesis A. Overview A T G C T G A C T A C T G T A C G A CT G A T G A C The protein product may be modified (have a sugar, lipid, nucleic acid, or another protein added) and/or spliced to become a functional protein. This is POST-TRANSLATIONAL MODIFICATION. (t-RNA) (r-RNA) U G C U G A C U A C U Amino acid (m-RNA) ribosome glycoprotein

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription a. The message is on one strand of the double helix - the sense strand: 3’ 5’ sense A C T A T A C G T A C A A A C G G T T A T A C T A C T T T T G A T A T G C A T G T T T G C C A A T A T G A T G A A A nonsense 5’ 3’ “TAG A CAT” message makes ‘sense’ “ATC T GTA” ‘nonsense’ limited by complementation

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription a. The message is on one strand of the double helix - the sense strand: 3’ 5’ sense A C T A T A C G T A C A A A C G G T T A T A C T A C T T T T G A T A T G C A T G T T T G C C A A T A T G A T G A A A nonsense 5’ 3’ exon intron exon In all eukaryotic genes and in some prokaryotic sequences, there are introns and exons. There may be multiple introns of varying length in a gene. Genes may be several thousand base-pairs long. This is a simplified example!

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription b. The cell 'reads' the correct strand based on the location of the promoter, the anti-parallel nature of the double helix, and the chemical limitations of the 'reading' enzyme, RNA Polymerase. Promoter 3’ 5’ sense A C T A T A C G T A C A A A C G G T T A T A C T A C T T T T G A T A T G C A T G T T T G C C A A T A T G A T G A A A nonsense 5’ 3’ exon intron exon Promoters have sequences recognized by the RNA Polymerase. They bind in particular orientation.

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription b. The cell 'reads' the correct strand based on the location of the promoter, the anti-parallel nature of the double helix, and the chemical limitations of the 'reading' enzyme, RNA Polymerase. Promoter 3’ 5’ sense A C T A T A C G T A C A A A C G G T T A T A C T A C T T T G C A U GUUU G C C A A U AUG A U G A T G A T A T G C A T G T T T G C C A A T A T G A T G A A A nonsense 5’ 3’ exon intron exon Strand separate RNA Polymerase can only synthesize RNA in a 5’3’ direction, so they only read the anti-parallel, 3’5’ strand (‘sense’ strand).

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription c. Transcription ends at a sequence called the 'terminator'. Promoter Terminator 3’ 5’ sense A C T A T A C G T A C A A A C G G T T A T A C T A C T T T G C A U GUUU G C C A A U AUG A U G A T G A T A T G C A T G T T T G C C A A T A T G A T G A A A nonsense 5’ 3’ exon intron exon Terminator sequences destabilize the RNA Polymerase and the enzyme decouples from the DNA, ending transcription

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription c. Transcription ends at a sequence called the 'terminator'. Promoter Terminator 3’ 5’ sense A C T A T A C G T A C A A A C G G T T A T A C T A C T T T G C A U GUUU G C C A A U AUG A U G A T G A T A T G C A T G T T T G C C A A T A T G A T G A A A nonsense 5’ 3’ exon intron exon Initial RNA PRODUCT:

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription c. Transcription ends at a sequence called the 'terminator'. Promoter Terminator 3’ 5’ sense A C T A T A C G T A C A A A C G G T T A T A C T A C T T T T G A T A T G C A T G T T T G C C A A T A T G A T G A A A nonsense 5’ 3’ intron exon G C A U GUUU G C C A A U AUG A U G A Initial RNA PRODUCT:

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing intron exon Initial RNA PRODUCT: G C A U GUUU G C C A A U AUG A U G A Introns are spliced out, and exons are spliced together. Sometimes these reactions are catalyzed by the intron, itself, or other catalytic RNA molecules called “ribozymes”.

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing intron exon Final RNA PRODUCT: AUG A G C A U GUUU G C C A A U U G A This final RNA may be complexed with proteins to form a ribosome (if it is r-RNA), or it may bind amino acids (if it is t-RNA), or it may be read by a ribosome, if it is m-RNA and a recipe for a protein.

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation a. m-RNA attaches to the ribosome at the 5' end. M-RNA: G C A U G U U U G C C A A U U G A

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation a. m-RNA attaches to the ribosome at the 5' end. M-RNA: G C A U G U U U G C C A A U U G A It then reads down the m-RNA, one base at a time, until an ‘AUG’ sequence (start codon) is positioned in the first reactive site.

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation a. m-RNA attaches to the ribosome at the 5' end. b. a specific t-RNA molecule, with a complementary UAC anti-codon sequence, binds to the m-RNA/ribosome complex. Meth M-RNA: G C A U G U U U G C C A A U U G A

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation a. m-RNA attaches to the ribosome at the 5' end. b. a specific t-RNA molecule, with a complementary UAC anti-codon sequence, binds to the m-RNA/ribosome complex. c. A second t-RNA-AA binds to the second site Phe Meth M-RNA: G C A U G U U U G C C A A U U G A

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation a. m-RNA attaches to the ribosome at the 5' end. b. a specific t-RNA molecule, with a complementary UAC anti-codon sequence, binds to the m-RNA/ribosome complex. c. A second t-RNA-AA binds to the second site d. Translocation reactions occur Meth Phe M-RNA: G C A U G U U U G C C A A U U G A The amino acids are bound and the ribosome moves 3-bases “downstream”

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation e. polymerization proceeds Ala Asn Meth Phe M-RNA: G C A U G U U U G C C A A U U G A The amino acids are bound and the ribosome moves 3-bases “downstream”

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation e. polymerization proceeds Asn Meth Phe Ala M-RNA: G C A U G U U U G C C A A U U G A The amino acids are bound and the ribosome moves 3-bases “downstream”

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation e. polymerization proceeds f. termination of translation Meth Phe Ala Asn M-RNA: G C A U G U U U G C C A A U U G A Some 3-base codon have no corresponding t-RNA. These are stop codons, because translocation does not add an amino acid; rather, it ends the chain.

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis 1. Transcription 2. Transcript Processing 3. Translation 4. Post-Translational Modifications Meth Phe Ala Asn Most initial proteins need to be modified to be functional. Most need to have the methionine cleaved off; others have sugar, lipids, nucleic acids, or other proteins are added.

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis C. Regulation of Protein Synthesis 1. Regulation of Transcription - DNA bound to histones can’t be accessed by RNA Polymerase - but the location of histones changes, making genes accessible (or inaccessible) Initially, the orange gene is “off”, and the green gene is “on” Now the orange gene is “on” and the green gene is “off”.

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis C. Regulation of Protein Synthesis 1. Regulation of Transcription Promoter 3’ 5’ sense A C T A T A C G T A C A A A C G G T T A T A C T A C T T T T G A T A T G C A T G T T T G C C A A T A T G A T G A A A nonsense 5’ 3’ exon intron exon RNA POLY Transcription factors can inhibit or encourage the binding of the RNA Polymerase. And, through signal transduction, environmental factors can influence the activity of these transcription factors. So cells can respond genetically to changes in their environment.

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis C. Regulation of Protein Synthesis 1. Regulation of Transcription 2. Transcript Processing Cut not made intron exon Initial RNA PRODUCT: G C A U GUUU G C C A A U AUG A U G A U A U A Mi-RNA’s and si-RNA’s are small RNA molecules that can bind to m-RNA and disrupt correct spicing, creating non-functional m-RNA’s.

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis C. Regulation of Protein Synthesis 1. Regulation of Transcription 2. Transcript Processing 3. Regulating Translation Meth Phe M-RNA: G C A U G U U U U G A A A U U G A Incorrect splicing can result in a ‘premature’ stop codon, terminating translation early, resulting in a non-functional protein.

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis C. Regulation of Protein Synthesis 1. Regulation of Transcription 2. Transcript Processing 3. Regulating Translation 4. Regulating Post-Translational Modification Meth Phe Ala Asn The patterns of cleavage and modification can vary.

VI. Protein Synthesis A. Overview B. The Process of Protein Synthesis C. Regulation of Protein Synthesis 1. Regulation of Transcription 2. Transcript Processing 3. Regulating Translation 4. Regulating Post-Translational Modification Affected by other cells Affected by other genes Affected by the environment Protein ? Gene activity is responsive to cellular and environmental cues

Cell Division A. The Cell Cycle INTERPHASE S G1 (DNA synthesis) Cytokinesis MITOTIC (M) PHASE Mitosis

A. The Cell Cycle     1.  Interphase:       a.  G1: high metabolic activity (protein synthesis) chromosomes diffuse; one DNA double helix per chromosome                

A. The Cell Cycle     1.  Interphase: Some cell types are "stuck" in this stage when they mature... it is only "stem cells" that keep dividing.  In some tissues, all stem cells  eventually mature, so the tissue can't regenerate (neurons)

LE 12-15 G0 G1 checkpoint G1 G1 If a cell receives a go-ahead signal at the G1 checkpoint, the cell continues on in the cell cycle. If a cell does not receive a go-ahead signal at the G1 checkpoint, the cell exits the cell cycle and goes into G0, a nondividing state.

Overview: Why Reproduce? A. The Cell Cycle 1. Interphase a. G1 b. S VII. CELL REPRODUCTION Overview: Why Reproduce? A. The Cell Cycle     1.  Interphase a. G1 b. S Chromosome duplication (including DNA synthesis) Centromere Sister chromatids

VII. CELL REPRODUCTION Overview: Why Reproduce? A. The Cell Cycle     1.  Interphase a. G1 b. S c. G2

VII. CELL REPRODUCTION Overview: Why Reproduce? A. The Cell Cycle     1.  Interphase 2. Mitosis

INTERPHASE PROPHASE PROMETAPHASE LE 12-6aa Centrosomes (with centriole pairs Chromatin (duplicated) Early mitotic spindle Aster Fragments of nuclear envelope Kinetochore Nonkinetochore microtubules Centromere Nucleus Nuclear envelope Plasma membrane Chromosome, consisting of two sister chromatids Kinetochore microtubule

METAPHASE ANAPHASE TELOPHASE LE 12-6ba Metaphase plate Cleavage furrow Nucleolus forming Nuclear envelope forming Centrosome at one spindle pole Daughter chromosomes Spindle

Overview: Why Reproduce? A. The Cell Cycle B. DNA Replication VII. CELL REPRODUCTION Overview: Why Reproduce? A. The Cell Cycle B. DNA Replication C. Mitosis G2 OF INTERPHASE PROPHASE PROMETAPHASE

Overview: Why Reproduce? A. The Cell Cycle B. DNA Replication LE 12-6da VII. CELL REPRODUCTION Overview: Why Reproduce? A. The Cell Cycle B. DNA Replication C. Mitosis   TELOPHASE AND CYTOKINESIS METAPHASE ANAPHASE

Overview: Why Reproduce? A. The Cell Cycle B. DNA Replication VII. CELL REPRODUCTION Overview: Why Reproduce? A. The Cell Cycle B. DNA Replication C. Mitosis   10 µm Nucleus Chromatin condensing Chromosomes Nucleolus Cell plate Prometaphase. We now see discrete chromosomes; each consists of two identical sister chromatids. Later in prometaphase, the nuclear envelope will fragment. Metaphase. The spindle is complete, and the chromosomes, attached to microtubules at their kinetochores, are all at the metaphase plate. Anaphase. The chromatids of each chromosome have separated, and the daughter chromosomes are moving to the ends of the cell as their kinetochore micro- tubules shorten. Prophase. The chromatin is condensing. The nucleolus is beginning to disappear. Although not yet visible in the micrograph, the mitotic spindle is starting to form. Telophase. Daughter nuclei are forming. Meanwhile, cytokinesis has started: The cell plate, which will divide the cytoplasm in two, is growing toward the perimeter of the parent cell.

Cleavage of an animal cell (SEM) Cleavage furrow Contractile ring of microfilaments Daughter cells Cleavage of an animal cell (SEM)

Cell plate formation in a plant cell (TEM) LE 12-9b Vesicles forming cell plate Wall of parent cell 1 µm Cell plate New cell wall Daughter cells Cell plate formation in a plant cell (TEM)