1	Biology Review Compounds and Bonds
2	Compounds A compound is a combination of elements. There are inorganic and organic compounds. Life is made up primarily of organic compounds. Organic compounds provide the richness and diversity that can embrace and embed the complexity and subtlety of the biological processes which we are about to discuss
3	Organic Compounds Because of carbon’s ability to form compounds with many other elements, the numbers of possible organic compounds is huge. Not only can a large number of specific organic compound exist, but they may exist in many isomeric forms, again enriching the choices by way of structural possibilities. Organic molecules also can repeat themselves in a polymeric fashion.
4	Chemical Bonds High-School Inorganic Chemistry has familiarized us with ionic bonds. Along with hydrogen bonding and van der Waals forces, the ionic bonds make up the class of non-covalent bonds. Covalent bonds are a different class of bonds, with a vast contribution to the structure of organic compounds.
5	Non-Covalent Bonds Among the non-covalent bonds are: · -ionic bonds. One electron maybe donated by one atom and accepted by the other.
6	Non-Covalent Bonds Among the non-covalent bonds are: · -hydrogen bonds: Relatively weak bonds formed between hydrogens participating in a dipolar covalent bond, such as fluorine, oxygen, nitrogen. The strongest hydrogen bonds are when the donor hydrogen atom and the acceptor are all in a straight line physically.
7	Non-Covalent Bonds
8	Covalent Bonds Covalent bonds are bonds between non-metals or between metal and non-metallic elements. Covalent bonds share an electron in the outer orbital. Among them are polar bonds between atoms with different atomic numbers non-polar bonds between atoms with approximately the same atomic number.
9	Covalent Bonds The elements of most interest in biology all have an incomplete outer atomic orbital shell. The outer orbital capacities are: Hydrogen 1 Carbon, Oxygen, Nitrogen, Phosphorous 8 Sulfur 12 The atom will share electrons (not necessarily all) with another atom with an incomplete outer shell. This is a covalent bond. The number of bonds varies with circumstances. Carbon usually has 4, hydrogen 1.
10	Covalent Bonds
11	Water Since non-polar molecules are not soluble in water, then water affects the molecular arrangement, by forcing non-polar moieties to aggregate together, while polar moieties want to be closer to the water. An example is salad oil in vinegar. Not only is the oil insoluble, it is forced into larger aggregates Another excellent example of this is 3 dimensional folding of proteins. Lecithin (egg yolk) is amphiphatic -acts as emulsifier
12
13	Genetic Theories Genetic Theories must account for How traits are passed/inherited from parents to offspring of individuals of the same species How those traits find expression in the individual
14	Genetic Theories We will account for the inheritance along with the fulfillment of the need for diversity using the Chromosomal Theory of Inheritance We will account for the expression of traits in an individual, or even in a cell, using the Central Dogma of Molecular Genetics
15	The Chromosomal Theory of Inheritance Traits are carried in in structures in the nucleus called chromosomes Every dividing cell, except sex cells, makes an exact copy of the chromosomes for the daughter cells. The sex cells diversify and rearrange the genetic material in the chromosomes. This rearranged genetic material can unite with a chromosome made in the same manner in a different individual of the same species.
16	The Central Dogma of Molecular Genetics-a Synopsis Genetic information is in DNA DNA is duplicated when a cell divides (Replication), so that each daughter cell has identical genetic information This genetic information is a set of blueprints for how proteins are to be built
17	The Central Dogma of Molecular Genetics-a Synopsis A copy of the blueprint for each protein is made from the DNA (Transcription) in the form of messenger RNA mRNA takes the blueprint and directs specific tools -transfer RNAs - to assemble the building blocks (amino acids) together, in the correct sequence, into a protein (Translation)
18	Contradiction? Chromosomal Theory of Inheritance vs Central Dogma of Molecular Genetics
19	Not really… The Chromosomal Theory tells us how both same-ness can be passed among cells in an individual and how diversity can generated for new individuals The Central Dogma tells us how the same-ness in the same individual and the diversity in the new individual come to be, i.e., how they are expressed
20	Chromosomal Theory To understand chromosomes in their modern context, we must understand that they have 2 key constituents: Protein DNA
21	Chromosomes Chromosomes are repositories for the master blueprint DNA is stored in Chromosomes When cells divide, the DNA in the chromosomes is replicated as the chromosomes ready themselves to send copies to the daughter cells
22	Expression and Regulation But before, during, and after cell division, the DNA continues to direct assembly of proteins (Expression) Which proteins, and at what rate, are also directed by the DNA in concert with a host of cellular environmental factors (Regulation).
23	Some Basic Biochemistry
24	What is DNA? Deoxyribonucleic acid is a polymer Each of the monomers consists of A nitrogenous base (one of 4 choices) deoxyribose (pentose [5 carbon ring] sugar) The monomers are linked together by a phosphate molecule intermediary
25	What is RNA? Ribonucleic acid is identical to DNA with the following two exceptions: Contains ribose instead of deoxyribose one of the carbons of the pentose is at a higher oxidation state One different nitrogenous base choice 3 bases are common to both DNA and RNA DNA has one unique base and RNA has one unique base
26	Other differences: In general RNA exists as a single polymeric strand It can double up on itself in certain instances DNA strand interacts with a second strand, forming a weak bond so the strands pair like RR tracks. This double stranded arrangement twists upon itself, forming an a-helix.
27	The Parts: Pentose sugars
28	The Parts: Nitrogenous Bases
29	Nucleosides When the base is attached to the pentose sugar, the structure is a nucleoside: adenosine guanosine cytidine, uridine (RNA only) deoxythymidine (DNA only)
30	The Parts: Phosphodiester
31	Nucleotides Adenyl triphosphate(ATP) Guanyl triphosphate(GTP) Cytidyl triphosphate(CTP) etc.
32	Nucleic Acid
33	Review of nomenclature For both DNA and RNA: A base + a ribose=nucleoside A nucleoside+phosphate ester=nucleotide A polynucleotide is a nucleic acid
34	We now know what DNA and RNA are… They are polymers of [(PENTOSE-BASE)-PHOSPHATE] units What about this double-stranded business?
35	Hydrogen Bonding: Base Pairing The Purine bases have a stereochemistry such that two or three stable hydrogen bonds can be made with a Pyrimidine. The Purine-Pyrimidine interaction is very specific: Adenine always (naturally) base pairs with Thymine. Anything else is a mutation Likewise, cytosine will always hydrogen-bind to guanine in both RNA and DNA.
36	Hydrogen Bonding: Base Pairing
37	Base Pairing
38	DNA Twists Into a Double-Helix The conformation of the paired polynucleotides, now weakly bonded together into a ladder-like arrangement, finds its minimal energy when the ladder twists into a helical structure, the B-DNA configuration, the double helix.
39	The Double Helix The helix takes one complete turn every 3.2 nanometers. The manner in which it coils leads to a minor groove and a major groove. It is right-handed; there are 10 base pairs per turn.
40	DNA -Double Helix Other stable forms of DNA exist. There is another right-handed helical structure that is similar, whose measurements are similar, called A-DNA There is left-handed structure with a tighter turn called Z-DNA.
41	RNA- Single Stranded RNA is single stranded, but m-RNA can certainly form a hybrid with a strand of DNA
42	RNA- Single Stranded
43	RNA- Single Stranded
44	Meanwhile, back to the Chromosomal Theory………..
45	Prokaryotes and Eukaryotes We can classify organisms into those which are a single cell with a simplified internal system and no nucleus ( the Prokarya) A bacterial cell is a good example of a prokaryote those with a complicated set of organelles and a well defined nucleus (the Eukarya) Any multicellular organism (including yeast) is an example of a eukaryote.
46	The Chromosomal Theory of Inheritance
47	Ploidy Humans have 24 distinctively sized/shaped chromosomes (22 autosomes, 2 sex chromosomes) Each autosome has another chromosome which roughly matches its size and shape – an homologous chromosome. Although the homologs are not quite identical, we say that they are a pair. The number of homologs is the ploidy. Humans are diploid.
48
49	Chromosomes
50	Chromosomes Become Distinct as DNA is Condensed Each chromosome has two arms connected together in a central spot, the centromere The telomeres are the ends of the arms.
51	Chromosomes The DNA strand length becomes compressed several thousandfold.
52	Chromosomes
53	Classical Inheritance somatic cells are diploid germ cells are haploid (one set of 22 autosomes and one X or one Y). These are the gametes, which will fuse with a gamete from a different individual during reproduction.
54	Classical Inheritance The mechanism of cell division differs between these two types. In a somatic cell, the idea is to make an exact copy of the parent’s genome In a germ cell, the idea is to set the stage for genetic diversity by Providing variation of the genetic material among individual cells Establishing haploid cells, so that after fusion the zygote can be diploid
55	Chromosomes-Mitosis Somatic Cells divide by mitosis Before the cell divides, each of the chromatids duplicates (DNA Replicates) During cell division, one chromatid from each homologous chromosome goes to one pole of the cell and the other pair of homologous chromatids to the opposite side. After the cell divides, the resultant child cells are diploid.
56	Chromosomes-Meiosis The situation is different in germ cell. Germ cells are haploid cells. These germ cells arise from somatic cells, which divide by a different mechanism, meiosis. The diploid cell undergoes a replication of the chromatids (DNA Replicates) but the chromatids do not separate and migrate; instead the two homologous chromosomes find each other and align with each other
57	Meiosis-Recombination 2. At this point an important phenomenon, crossing over and recombination take place. 3. Sections of one chromatid break and reunite with their counterparts on the homologous chromatid
58	Meiosis First Division 4. Chromosomes, which have undergone crossing over, move to opposite sides of the cell. In mitosis the chromatids separate at this stage; in meiosis they don’t.
59	Meiosis 5. After the cell divides in the first meiotic division, one chromosome ends up in each child’s cell. 6. There is a second cell division, during which the chromatids separate, one for each new cell. 7. After this second meiotic division, the 4 daughter cells of course are now haploid, as one would expect a gamete to be.
60	Mitosis vis à vis Meiosis To summarize this important distinction: The idea of mitosis is to make an exact copy of the genome in a somatic cell. The idea of meiosis (only in germ cells) is to make a combination, rather than an exact copy, and to distribute these ‘mixed’ strands to different cells in a haploid form, suitable for eventual fusion with an external gamete, setting the stage for genetic diversity.
61	Chromosomal Theory of Inheritance If we envision ‘chunks’ of these chromosome as carrying traits, and if we very loosely associate the word gene to these traits, we have the chromosomal theory of inheritance. In this theory, corresponding genes from the two chromosomal homologs have a relationship characterized as either co-dominant or dominant-recessive.
62	Alleles In classical experiments with peas, traits are easy; they could be ‘yellow’ or ‘green’. Yellow and green are the expression of the alleles of the color gene. An allele is one of the choices of expression of a gene Pea color is a good example; yellow is dominant over green. If one chromosome has the yellow allele and the homologous chromosome has the green allele, the pea will be yellow
63	Multiple Alleles We well know that there are many situations where a trait is not a simple dichotomy involving only 2 alleles. Examples of multiple allelism are eye color and ABO blood type system. In eye color there are multiple alleles, but brown is dominant. In blood types, there are the alleles A,B and O. O is always recessive
64	"Eukaryotic chromosomes contain DNA" Eukaryotic chromosomes contain DNA The chromosome is made up of 50% protein and 50% DNA. The genomic DNA is broken up into 46 pieces Of the 3 billion base pairs in the human genome, each of the 23 chromosomes contain strands of DNA with between 120-300 million of these bases, and its homologous chromosome likewise. There is only one DNA molecule in each chromosome
65	The Central Dogma Subsequent to the characterization of DNA in the 1950’s by Watson and Crick, the understanding of genetics at the molecular level has advanced exponentially. The advances occupy many volumes of many textbooks, and increase daily. The entire theory of molecular genetics, however, can be summarized by a very succinct and robust set of principles suggested by Watson and Crick:
66	The Central Dogma of Molecular Genetics The very linchpin of the “Central Dogma” is the fact that DNA is the fundamental vehicle of inheritance. DNA is replicated during cell division. Replication only occurs during cell division Parts of the DNA are transcribed into messages. The message is encoded in RNA . The message in the RNA is then translated into a blueprint for protein synthesis. Transcription and translation are going on all the time
67	The Central Dogma of Molecular Genetics Things to think about: All information is in the DNA. It gets there by inheritance and by mutation (and by infection). The cellular proteins can regulate DNA and RNA expression at many levels.
68	Enzymes to Facilitate Replication, Transcription,Translation Polymerases DNA Poymerase duplicates a single strand of DNA RNA Polymerase makes a complementary copy of a single DNA strand Ligases: DNA ligases connect two contiguous fragments through a diester bond. Endonucleases: Break the phosphate linkage, cleaving a strand into two parts.
69	Important Polymerase Details DNA polymerase attaches nucleotides to each other, stepwise, always from the 5’ to the 3’ end, by removing 2 phosphates and joining the sugars through the remaining phosphate in a phospho-diester bond. DNA polymerase never initiates poymerization de novo; it must always have a piece of material to builds upon—a primer. This primer may be either DNA or RNA. RNA polymerase likewise attaches nucleotides to each other 5’ to 3’. It recognizes the difference in bases between DNA and RNA (ie the uracil). RNA polymerase does not require a primer; it may initiate de novo synthesis.
70	Endonuclease Details There are many endonucleases which differ in the 4 or 5 base pair context in which they work. For example, the enzyme EcoRI from E. coli recognizes a specific context G¯AATTC. Both strands are cut, so the same context must be running in the opposite direction on the other strand. If the cleavage sites are staggered, then there are free single stranded ends, called sticky ends; if they are coincident, the cleavage is said to be blunt. Endonucleases with specificity of this nature are called restriction endonucleases, or restriction enzymes.
71	Replication The short version: The double-strand separates sequentially, like un-zipping. If the constituent nucleotide building blocks are present, then DNA polymerase will form a strand which is a complimentary copy of each base that it sees.
72	Replication-Details The longer version: Helicase unwinds the helix. As it unwinds, the torsion on the strands cause them to form coils. Isotopomerase relaxes the consequent supercoiling. .
73	DNA Replication-Details Now, to copy the single strands, there are two problems: DNA polymerase needs a primer; it cannot copy de novo. While the leading strand can be copied 5’ to 3’ toward the fork, the lagging strand would need to be copied backwards (3’ to 5’).
74	DNA Replication-Details The solutions to these problems are a bit complicated. The primer problem is solved by a special form of RNA polymerase, which builds a short segment of RNA complementary to the DNA template. DNA polymerase treats this segment as a primer and goes on to extend the primer, sequentially adding nucleotides to the strand which are complementary to the nucleotides in the template strand. The lagging strand problem is also solved by a special RNA polymerase. Every 1000 nucleotide positions or so, along the unzipped single lagging strand, a short segment of RNA is deposited. This RNA acts as a primer for the DNA polymerase to extend until the next island of RNA is encountered. At that point the RNA primer is removed, and the DNA continues its extension until it hits DNA. DNA ligase then joins the extending DNA to the adjacent piece. Pretty magic, huh?
75	DNA Replication-Growing Fork
76	Transcription The Central Dogma applies to eukaryotes and prokaryotes alike. That said, there are some fundamental differences in transcription. We begin with a definition of a gene, here taken from Lodish, Berk et al.: “A gene is a unit of DNA that contains the information to specify synthesis of a single polypeptide chain.”
77	Prokaryotic Transcription In prokayotes, genes which in concert accomplish some metabolic task, are found in contiguity in the DNA. The collection of commonly oriented genes is called an operon. The operon is transcribed sequentially. The messenger, messenger RNA (mRNA), carries the entire operon message in an uninterrupted strand.
78	Eukaryotic Transcription
79	Transcription Prokaryote vs Eukaryote
80	Exons and Introns In eukaryotes, genes are not necessarily in contiguity; in fact, they almost never are. The coding parts of genes, called exons, are interspersed with non coding parts, introns.
81	"Prokaryotes have no introns" Prokaryotes have no introns. 90% of the DNA is coding In eukaryotes , the introns, together with non coding sequences, account for most of the DNA. Only 3 % is coding from exons.
82	Eukaryotic Transcription Complicating Features Because There Are Introns Conceptually, to form mRNA for a gene, it might be necessary to read parts of a gene, skip over non-relevant material in the transcription process, then continue with relevant gene information.
83	Transcription Troubles… The mRNA polymerase must know where to start reading DNA at the beginning of a gene The mRNA itself must know where to ignore introns and ‘connect’ exons Certain subsequences of bases give broad hints about these events. Such hints are called signals
84	Transcription Signaling The regions of DNA that herald the start of a gene are called promoter signals The signals in mRNA that mark the beginning and end of introns are called splicing signals
85	Eukaryotic Transcription Promoters A promoter is a DNA sequence which signals where transcription will begin. TATA Box: TATA^T/_AA at -25 CpG Islands: C,G rich 20-50 long stretch at –100
86	Eukaryotic Transcription-Example RNA Polymerase II binds to a specific starting place on the DNA.
87	Eukaryotic Transcription Here’s how a gene is transcribed: RNA polymerase transcribes from some starting point relative to a start signal and continues until it reaches a stop signal The mRNA transcript is ‘protected’ at each end by a ‘wrapper’ A cap at the 5¢ end A tail at the 3¢ end
88	Eukaryotic Transcription The 5’ end of the primary transcript is marked and protected with a cap, a two phosphate ester from the 5’ position of the end nucleotide to the 5’ position of a methylated guanylate The first nucleotide ( and in vertebrates the second nucleotide as well) are methylated on the 2’ position.
89	Eukaryotic Transcription At the 3’ end, an endonuclease makes a 3’ hydroxyl group to which 100-250 (depending on the species) copies of adenylic acid are added. The RNA polymerase for this is special: Poly(A) polymerase.
90	SPLICING
91	Eukaryotic Transcription:Splicing How does it know…? The intron always has a GU at the 5-prime boundary and AG at the 3-prime boundary. Evidently these are sine quae non; if those sequences are altered, then the whole splicing sequence with its precision is altered
92	Something to think about…… One the gene is spliced, the mRNA has the complement of the ‘pure exon’ gene. DNA made from that mRNA would be a non-genomic, ‘pure exon’ DNA This is done in real life. It is called complementary DNA or cDNA. cDNA is the cornerstone of gene-chip technology.
93	Details on Eukaryotic Transcription:Splicing The splicing reaction takes place in the nucleus in the eukaryotes and is not totally understood, but seems to involve the following parts: small nuclear Ribonuclear Proteins (snRNP’s) , which evidently have miniature templates for commonly occurring gene motifs a lightweight particle called spliceosome that seems to be involved in coordinating the un-spliced messenger RNA this snRNP’s and some how mediating the splicing. the spliced out introns are consumed immediately.
94	Genefinding Implications Splicing is a big deal when teaching machines how to recognize genes. Learning models must understand how to distinguish between introns and exons
95	Translation The message in mRNA is a template which will direct the synthesis of a protein from amino acids. The structural “machine” in which the synthesis takes place is the ribosome. It is made of special RNA (rRNA) and protein
96	Translation
97	RNA- Roles inTranslation RNA thus has two roles in translation: Ribosomal RNA ( rRNA) is a structural element of the ribosome – Transfer RNA (tRNA) is the carrier which brings an amino acid to the ribosome, then attaches it in the sequence specified by the mRNA
98	Translation Transfer RNA has a structure which resembles a Celtic cross turned upside-down. In this allegory, the shaft is the 3-prime end of the single stranded RNA chain, which is folded on itself to make the cross. This always ends in ACCA-OH; that is where the amino acid is attached - the acceptor end.
99
100	Translation
101	AntiCodons on tRNA read Codons on mRNA
102	The Genetic Code There are 20 amino acids that are coded for, although others may be created in post-translational processing. Since there are 4 possible bases, taking 3 at a time, there are 4³ or 64 codons, resulting in a highly degenerate system.
103	The Genetic Code The cDNA equivalent uses a T instead of U
104	The Genetic Code There are 3 dedicated codons to mark the end of the gene, the stop codons: UAA, UAG, UGA. The mRNA always attaches to the ribosome at AUG, the start code( methionine). The code is degenerate: arginine, leucine and serine each have 6 codons. Methionine and tryptophan , the least common amino acids, have single codon. The rest of the amino acids have 2, 3, or 4 codons. Most of the synonyms, that is, different codons which code for the same amino acid, differ only in their third nucleotide (with the exception of arginine, leucine, and serine).
105	"DNA and mRNA are interpreted..." DNA and mRNA are interpreted by three’s; three nucleotides in succession make a codon One can easily envision difficulty in knowing where to start, exactly, in transcribing DNA. The sequence of bases taken from a particular start point is called a reading frame. From the 5-prime to the 3-prime end, there are 3 reading frames.
106
107	Reading Frames The reading frame idea can really backfire if: Reading starts in the wrong place There is a deletion, throwing off the count There is a transposition, not registering modulo 3 The translation can, among other things, Be gibberish Miscode into a mutation Keep right on going past the poly-A (polylysine) tail