bio unit unit 3 Molecular genetics Flashcards
(93 cards)
1
Q
Discovery of the existence of DNA
A
- DNA was discovered in 1869, by Swiss chemist, Friedrich Miescher–Although it was not known as genetic material
- He isolated the nuclei of white blood cells from pus-soiled bandages
- It contained nitrogen and phosphorous
- He named it Nuclein
- Renamed to Nucleic acid
2
Q
Phoebus Leven
A
- In the early 1900’s Phoebus Levene isolated two types of nucleic acid (now called DNA and RNA)
- In 1919 he proposed that DNA and RNA are polymers made up of single units (monomers) called nucleotides
- Containing four N-containing bases, a sugar molecule, and a phosphate group
- DNA is a polymer of nucleotides (not sure of the shape)
- DNA has nucleotides consisting of 4 different nitrogenous bases: adenine (A), thymine (T), cytosine (C), guanine (G)
- RNA has the same nitrogenous bases, except, thymine (T) is replaced by uracil (U)
- each of DNA’s four types of nucleotides consists of Deoxyribose sugar (5-C) attached to a phosphate group and a nitrogenous base
- Recall - the carbon atoms are numbered clockwise, starting with the carbon atom to the immediate right of the oxygen atom. The first carbon is called 1′ (1prime), followed by 2′ (2 prime) and so on
2
Q
By the 1900’s, we assumed/knew
A
- Knew about inheritance of traits
- Assumed it was linked to chromosomes
- Knew chromosomes were composed of nucleic acids and proteins
- They assumed proteins within chromosomes carried the hereditary genetic material that caused the inheritance of traits
3
Q
Griffith’s Discovery of Transformation
A
- “The Transforming Principle” –In 1928, Frederick Griffith studied the pathology (disease causing characteristics) of the bacteria Streptococcus pneuomoniae
- He studied 2 strains of bacterium in mice:
1) Disease causing S form
2) Harmless R form - Inject mice with R (nonvirulent) cells and the mice lived.
- Inject mice with S (virulent) cells and the mice died. Blood samples from the dead mice contained many S cells.
- S cells were killed with heat, then injected into mice and the mice lived.
- R cells plus heat-killed S cells were injected into mice and the mice died. Living S cells were found in the blood
- The conclusion was that some unknown substance from the dead S cells had transformed the harmless R cells into cells capable of causing death.
- Descendants of the transformed cells were also pathogenic (to cause disease).
- Discovered the process of transformation
- TRANSFORMATION: The introduction of foreign DNA, usually by a plasmid or virus, into a cell.
- Griffith transformed nonvirulent pneumococcus into virulent pneumococcus with S-strain DNA
3
Q
TRANSFORMATION
A
The introduction of foreign DNA, usually by a plasmid or virus, into a cell.
4
Q
Chargaff’s Rule
A
- Late 1940’s: Austrian-American biochemist Erwin Chargaff studied and compared DNA from different species.
- He observed that nucleotides and their nitrogenous bases are always present in characteristic proportions
- Example: –the amount of adenine in any sample of DNA is always approximately equal to thymine
- the amount of cytosine is always approximately equal to the amount of guanine
- This constant relationship is known as Chargaff’s Rule
4
Q
Chase and Hershey
A
- Experiment involved infecting a bacteria using a virus (bacteriophage) which consisted of two components: DNA and a protein coat
- They knew that viruses replicated themselves by inserting their own hereditary material into host cells, so they were attempting to isolate what the hereditary material was that the viruses were inserting
- In the Hershey–Chase experiment, radioactive phosphorus in viral DNA and radioactive sulfur in viral proteins were used to trace the transfer of each type of biological molecule into a bacterial host cell
- One of the most famous experiments in history of genetics
- Ruled out protein in favour of DNA as the hereditary material
- Experiments showed that only the DNA and not the protein coat entered the cell (labelled DNA with P isotope and protein with S isotope).
- Hershey and Chase’s experiment clearly showed that DNA is the hereditary material
4
Q
Avery, Macleod and McCarty
A
- By 1944 researchers could grow bacteria in liquid cultures
- They prepared cultures of heat-killed S-strain bacteria
- They added 1 of 3 enzymes to each bacteria (Enzyme destroying proteins, Enzyme destroying RNA, and Enzyme destroying DNA)
- When treating the non-virulent R-strain with these modified heat-killed S-strains, the only enzyme-treated bacteria that did not create a virulent R-strain was the DNA-destroyed bacteria
5
Q
Linus Pauling
A
- Developed methods of assembling three-dimensional models based on known distances and bond angles between atoms in molecules
- This helped him discover that many proteins have helix-shaped structure
6
Q
By the late 1940’s scientists knew
A
- From Hershey & Chase: DNA is the hereditary material
- From Levene: DNA is a polymer of nucleotides and nucleotides have different nitrogenous bases (A,T,G,C)
- From Chargaff: DNA is composed of these nucleotides that exist in fixed proportions (A=T, G=C)
7
Q
Rosalind Franklin
A
- Used x-ray diffraction to analyze structure of biological molecules –Was able to obtain highest resolution photographs at that time
- Based on her images, was able to conclude that DNA has a defined helical structure and had two regularly repeating patterns
- When DNA reacted with water, she concluded that nitrogenous bases were located inside of helical structure, and sugar-phosphate backbone was located on outside of helical structure
8
Q
Watson & Crick (1953)
A
- Proceeded to construct the current accepted molecular structure for DNA
- Watson & Crick concluded that DNA has a twisted, ladder-like structure called a double helix
- Sugar-phosphate molecules make up sides or “handrails” of ladder
- Bases make up the rings
8
Q
Modern DNA Model: the Double Helix
A
- Two polynucleotide strands that twist around each other forming a double helix
- Complementary Base Pairing: A-T, C-G
- Hydrogen Bonds link complementary base pairs
- A and T share 2 hydrogen bonds
- C and G share 3 hydrogen bonds
- Two strands of DNA are antiparallel
- One strand runs in the 5′ to 3′ direction and the other strand runs in the 3′ to 5′ direction
- Sugar-phosphate backbone
- Contains Major and Minor grooves (not symmetrical)
- 5’ end finishes with a phosphate group sticking up
- 3’ end finishes with a hydroxyl group pointing down
- In RNA, the 2’ Carbon has a hydroxyl (OH) group attached instead of a H
9
Q
Antiparallel
A
the two strands of DNA run in the opposite direction
10
Q
Building DNA polymers
A
Each strand is made from the bonding of a phosphate group of one nucleotide with the #3 carbon of the next nucleotide via a phosphodiester linkage
11
Q
Genes
A
- the basic unit of heredity that determines, in whole or part, a genetic trait;
- a specific sequence of DNA that encodes for proteins and RNA molecules, and can contain sequences that influence production of these molecules.
- A gene is a specific chain of base pairs that form specific proteins. They are always found together in that order
- The space between genes is variable, and may change as it is passed down with little effect on the protein formed
12
Q
Genome
A
the complete genetic makeup of an organism; an organism’s total DNA sequence
13
Q
DNA Regulation
A
- Regulatory sequence (turning genes on and off)– a sequence of DNA where proteins bind and regulate the activity of a gene (inhibiting or activating it)
14
Q
Nucleoid
A
the structure that contains the chromosomal DNA.
15
Q
DNA supercoiling
A
The formation of additional coils in the structure of DNA due to twisting forces on the molecule.
DNA Supercoiling in Eukaryotic Cells
- Histones – a member of a family of proteins that associate with DNA in eukaryotic cells, which acts to help compact the DNA
- Nucleosome – the condensed structure formed when double stranded DNA wraps around an octamer of histone proteins (8).
- first, DNA wraps around histones, which are protein balls. When 8 histones come together, they become a nucleosome (is an octomer) that also has DNA wrapped around it. The DNA wrapped nucleosomes form euchromatin, and then heterochromatin, and then chromosomes (Theres 23 pairs in somatic). This makes the DNA very compacted. This means that this process happens 46 times in somatic cells and 23 times in sex cells
- Also, little DNA does NOT mean the organism isn’t complex
16
Q
DNA of Prokaryotic Cells
A
- In bacteria, the amount of supercoiling is controlled by two enzymes: topoisomerase I and topoisomerase II.
- Additional proteins help stabilize the fold
- topoisomerase II – enzyme is essential for bacterial survival
- Antibacterial drugs have been developed that specifically target and block activities of this enzymes.
- Ex. Quinolones and coumarins
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Q
Plasmids
A
one or more small circular or linear DNA molecules. These tend to carry non-essential genes and can be transferred and copied from cell to cell.
18
Q
DNA of Eukaryotic Cells
A
- The total amount of DNA is much greater
- Genetics material is located in the membrane bound nucleus
- The approximate length of DNA in the nucleus of a single human cell is 2 meters long
- a nucleus is 4 micrometers wide.
- Most eukaryotes are diploid – they contain two copies of each chromosome/gene.
- Some eukaryotes are haploids, such as ferns and algae
- The organization of genes on each chromosome can differ
- Chromosome 19 has 72 million base pairs and 1450 genes
- Chromosome 4 has almost 1.3 billion base pairs and about 200 genes.
- There is no correlation between an organisms complexity and genome size or number of protein-coding genes
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Q
Chromatin
A
- Additional compacting of DNA occurs and becomes chromatin
- non-condensed form of genetic material that consists of a complex of DNA and proteins
- euchromatin (compacted) FIRST
- heterochromatin (highly compacted) SECOND
- after chromatin is chromosome
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Life Cycle of a Cell
- The process of copying one DNA molecule into two identical molecules is called DNA replication
- Occurs during the S phase of interphase in the cell cycle
- All cells must reproduce before they die to pass on genetic information. Since they will live for a little bit longer passed cell division, they will need a copy of their own DNA.
In order for cells to divide they must:
- Grow
- Carry out metabolic activity
- Replicate DNA
- During division, each daughter cell contains exact same genetic material as parent cell
- DNA Replication: process of producing two identical DNA molecules from an original, parent DNA
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Three Proposed models of DNA Replication
- The conservative model results in one new molecule and conserves the old.
- The semi-conservative model results in two hybrid molecules of old and new strands.
- The dispersive model results in hybrid molecules with each strand being a mixture of old and new strands.
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Meselson and Stahl Experiments on DNA Replication
- Through experimentation showed that semi-conservative model of DNA replication was correct
- Used two different isotopes of nitrogen to distinguish between parental and daughter DNA strands
- Used different isotopes of Nitrogen to label DNA in a cell
- 14N (common, lighter) and 15N (rare, aka “heavy”, when decaying, will become 14N)
- Used nitrogen because it's found in all of our nitrogenous bases
- after spinning all of the nitrogen 15, they put it in the nitrogen 14 medium
- they saw that 15N was still there, but new strands were made
- They replicated again and noticed 14N was appearing more than N15.
- Since N14 was more than N15, this helped us to know DNA was semi-conservative
- If it was conservative, N15 would be the same the whole time
- If dispersive, both N15 and N14 would be equal
- At first, their experiment was only planned for N15, but then they switched it to N14
Starting the experiment:
- They grew E. coli bacteria in a medium with 15N for 17 generations
- By doing this, the E. coli could only replicate DNA using the heavy 15Nitrogen Isotope within their nitrogenous bases
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Initiation
Portion of the DNA double helix is unwound to expose the bases for new base pairing
DNA Replication - Initiation
- During S-phase during interphase
- Replication starts at a specific nucleotide sequence called The Origin of Replication
- DNA helicase unwinds the double helix by breaking the H bonds between the complementary base pairs holding the two DNA strands together. It has a hole which has things like teeth that unwind the DNA, unzips DNA. For example, the triple bond between G and C will be broken by helicase
- Behind helicase is the replication fork,
- Single stranded binding proteins (SSBs) keep the individual strands apart by blocking the hydrogen bonding between the bases. Since the strands want to be helicase structure, SSBs help each strand to make the hydrogen bonds not connect
- Topoisomerase II (also called Gyrase)– relieves stress of the unwinding on the parent DNA molecule by cutting and un-twisting the molecule. When unwinding, the ends of the DNA strands will supercoil, which will cause it to break. Can be solved by having gyrase to relieve it by cutting the DNA, usually binds at the ends
- Replication starts at a specific nucleotide sequence - the origin of replication
- As the two strands of DNA are disrupted, the junction where they are still joined is called the replication fork
- In eukaryotes, DNA replication occurs at more than one site at a time, resulting in hundreds of replication forks across a DNA strand. This is important because it speeds up the process.
- When 2 replication forks form, a replication bubble is also formed
- If the helicase is seen on the left, DNA is unziping on the left and is headed towards the left direction, INTO THE FORK
Transcription- mRNA initiation
- DNA transcription only occurs on one strand: the template strand
- There is no need to transcribe from the coding strand because it is identical to the mRNA being formed (except it has Thymine not Uracil)
- RNA polymerase: The main enzyme that catalyzes the formation of RNA from DNA
- DNA is unwound by RNA Polymerase to expose the template strand
- Also remember that the template strand must be 3’-5’. This allows the mRNA to be built 5’-3
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Semi-Conservative Replication
- Mechanism of DNA replication that produces two copies
- both are made up of one new strand and one conserved from original DNA
There are 3 basics phases in replication:
- Initiation
- Elongation
- Termination
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Elongation
- Two new strands of DNA are assembled using parent DNA as template
- New DNA molecules (each composed of one strand of parent DNA and one strand of daughter DNA) reform into double helices
Process of DNA Replication - Elongation
- DNA polymerase III - main player that adds nucleotides to the new strand of DNA. can only add nucleotides in the 5′ to 3′ direction, and require RNA primase as starting points, and requires condensation reactions to go through the 5' to 3' direction. RNA primers are the starting site because DNA polymerase III will immediately know that it should combine to it
- DNA is always synthesized in the 5′ to 3′ direction
- The leading strand is built continuously by Polymerase III toward the replication fork, starting with 1 RNA primer
- The lagging strand is synthesized by Polymerase III discontinuously in short fragments in the opposite direction to the replication fork
- These short fragments are called Okazaki fragments which each require RNA primers
- The enzyme Primase lays down RNA primers that will be used by DNA polymerase III as a starting point to build the new complementary strands
Why do we need RNA Primers?
- Allows DNA Polymerase III to bind to the strand
- DNA polymerase can only add new nucleotides to a free 3′ end of a growing chain of DNA
- DNA polymerase I removes the RNA primers from the leading strand and from the lagging strand’s fragments. It will then fill in the space with DNA nucleotides by extending the neighbouring DNA fragment
- DNA ligase enzyme joins the Lagging strand’s Okazaki fragments into one strand (if making RNA, then it's RNA ligase)
- Synthesis of one strand of DNA (leading strand) proceeds continuously in the 5′ to 3′ direction
- Synthesis of the complementary strand (lagging strand) is more complex because it is running opposite to the leading strand, and DNA polymerase can ONLY add new nucleotides to a free 3′ end
- To solve this dilemma, the polymerase builds the lagging strand using many small pieces called Okazaki fragments
- DNA polymerase I will come and remove the RNA primers at the end of elogation
- On the leading strand, there's only 1 RNA primer. If a bubble, there's 2 on it.
- Primers are always on the 5' end of the newly synsesized DNA
Transcription- mRNA
- RNA polymerase reads the template strand and adds complimentary RNA nucleotides in the 5’-3’ direction
- Thymine (T) is replaced by Uracil (U)
- No Okazaki fragments form
- As soon as this begins, another RNA polymerase can bind to the promoter region and start building another strand of RNA (rapid production of RNA)
- RNA Polymerase can synthesize new strands much faster than DNA Polymerase could during DNA Replication
- RNA Polymerase does not proofread the RNA! This is because when there's a mistake, the RNA turns to protein unlike DNA, which would cause the cell to die if there's a mistake
- During elongation, an RNA polymerase complex moves along the DNA strand, the DNA helix unwinds, and complementary RNA nucleotides are joined together. After the RNA polymerase has passed, the DNA double helix reforms.
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The Lagging Strand
- RNA primase attaches to DNA and synthesizes a short RNA primer (makes an RNA primer, a sequence of about 10 nucleotides, complementary to the parent DNA)
- DNA polymerase III then adds nucleotides to the 3′end of the RNA primer
- DNA polymerase I comes in and removes the RNA primers and replaces it with DNA
- DNA ligase forms a phosphodiester bond between the 3′ OH of the growing strand and the 5′ phosphate in front of it
- DNA is further unwound, new primers are made and DNA polymerase III jumps ahead to synthesize another Okazaki fragment
- Synthesized discontinuously
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Termination
DNA Replication
- Replication process is completed
- Two new DNA molecules separate from each other
- Replication machine is dismantled
- Occurs upon completion of the new DNA strands
- New DNA molecules separate from each other
- Replication machine is dismantled
- Everything falls off (helicase, DNA polymerase III, etc.)
- The three DNA polymerase proof read the nucleotides
- While elongating DNA, polymerase III proof reads DNA (initial proofread)
- When polymerase I removes RNA primers to add DNA, the second proofreading happens
- The third time happens when DNA polymerase II is at the end to proofread
Transcription- mRNA
- Specific sequence signals the end – STOP sequence
- When RNA polymerases reach this sequence, they detach
- The newly synthesized RNA is released and ready to be processed into mRNA
DNA double helix reforms
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Forming mRNA
- Recall: ALL nucleic acids are synthesized 5’to 3’
- Every new nucleotide is added to a free 3’ –OH group
- mRNA is synthesized off of the 3’ to 5’ DNA Strand
- The template (antisense) strand is the 3’to 5’ strand of DNA
- This is also the leading strand in DNA replication
- mRNA is the same sequence as the 5’ to 3’ DNA strand, with U instead of T
- the coding (sense) strand is the 5’ to 3’ strand of DNA
- Also known as the lagging strand in DNA replication
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Topoisomerase 2
Releases strain on the parent DNA molecule due to the unwinding process ahead of any replication forks.
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Correcting Errors
- DNA Polymerase I and II proofread newly synthesized DNA
- DNA polymerases remove incorrect bases
- Mismatch repair involves proteins recognizing mispaired nucleotides and replacing them
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DNA
- DNA stands for deoxyribonucleic acid
- DNA controls all the chemical changes which take place in cells
- The kind of cell which is formed, (muscle, blood, nerve etc) is controlled by DNA
- Before a cell divides, the DNA strands unwind and separate
- Each template strand allows DNA Polymerase III to add a new strand by adding the appropriate nucleotides
- Result is that there are now two double-stranded DNA molecules in the nucleus
- When cell divides, each nucleus contains identical DNA
- This process is called replication
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The link between DNA and Proteins
- In 1953, Frederick Sanger showed that proteins consist of amino acids and that each protein consisted of specific amino acid sequences.
- By the 1960’s a clear link was formed between genes and proteins
- BUT how information was going from DNA to Proteins of amino acids was a still a mystery
- RNA is the link between the two
- RNA is slightly more stable than DNA because it has a OH group on the second carbon of its sugar
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RNA
- A genetic Messenger between DNA & Protein
- Scientists began to look at RNA as an intermediary between DNA and proteins (Jacob and Monod, 1961)
- Ribonucleic acid (RNA) is a single stranded polynucleotide.
- 2’ OH group on ribose sugar (DNA has H)
- Nitrogenous base pair of uracil (U) instead of thymine (T)
- RNA is synthesized in nucleus and transported to cytoplasm
- This keeps the chromosome in the nucleus, safe from harm
- The mRNA will also get degraded like DNA, but not as fast, and can make many proteins before degrading
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mRNA
- messenger
- RNA that contains the genetic information of a gene and carries it to the protein synthesis machinery in the cytoplasm
- mRNA is complimentary to the gene DNA sequence, opposites that complete each other
- template strand: 3' to 5'
- coding strand is the same as mRNA strand, except T is coding turn to U in mRNA, both 5' to 3'
- IMPORTANT: body uses template strand to make the complimentary mRNA strand
- mRNA holds the instructions for building chains of amino acids that will form proteins
- In 1964, Francois Jacob and Sydney Brenner made these conclusions through experimentation:
-When bacteria were infected by a virus, a virus specific RNA molecule was synthesized and became associated with pre-existing bacterial ribosomes.
-The new RNA molecule has a base sequence complimentary to the DNA and carried the genetic information to produce the viral protein
-The viral RNA molecule was newly synthesized and was not a permanent part of the bacterial ribosomes
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other types of RNA
- tRNA (transfer RNA)
- rRNA (ribosomal RNA)
- snRNA* (small nuclear RNA)
- siRNA* (small interfering RNA)
- crRNA (CRISPR RNA)
CRISPR gathered lots of attention in the past few years. crRNA can be created by humans to match to a specific region of DNA, and with the protein Cas9, remove it and replace it with DNA of our choosing – Gene Editing (cutting edge technology) Possible key to replacing antibiotics.
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Genetic Code
- The sequence of bases in DNA forms the Genetic Code
- A set of rules for determining how genetic information in the form of a nucleotide sequence is converted to an amino acid sequence of a protein
- There are 4 nucleotides and 20 amino acids
- Nucleotide bases of mRNA are read 3 at a time, this is known as the triplet hypothesis
- Each triplet is called a codon (e.g. GCU or UUU)
- There are 64 different codons (43 = 64)
- Combinations = possible choices to the power of the number of places in a codon
- mRNA is read as Codons of 3. Each codon will code for an Amino Acid
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Reading mRNA Codons as Amino Acids
- Start by finding the first letter of the mRNA codon in the “first base” column.
- Read across the rows in the “second base” column to find the second letter of the codon. This will take you to four possible amino acids
- Finally, read down the “third base” column to find the last letter of the codon.
- The last letter of the codon combined with the previous two letters, identifies the amino acid that corresponds to the codon
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Characteristics of Genetic Code
It is redundant
- More than one codon codes for same amino acid
- E.g. AUU and AUC code for isoleucine
There are 3 codons that do not code for an amino acid
- They are called STOP Codons (end of translation)
- UAA, UAG, UGA
- It is continuous (no spaces, no overlap)
- It is universal (except for some protists)
- AUG is the START codon (methionine)
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DNA and Enzymes
- DNA allows for proteins to be constructed, proteins build cell structures
- Proteins can also be enzymes
- The DNA controls which enzymes are made, and the enzymes determine what reactions take place
- The structures and reactions in the cell determine what kind of a cell it is and what its function is (muscle cell, blood cell, bone cell, sperm cell, nerve cell etc.) 200 Different cell types in the human body!
- DNA exerts its control of a cell through the enzymes it codes for
- A sequence of triplets in the DNA molecule may code for 1 complete protein
- Such a sequence forms a gene
- There may be a thousand or more bases in one gene
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The Central Dogma of Genetics
- A rule or set of rules known to be irrevocably true
-so true and known to be, that it will not be changed in the future
- DNA is too valuable to leave the nucleus
- It could be damaged, leading to cell death
- mRNA carries genetic info out of nucleus
- Genes on DNA are regulated, allowing for mRNA to be synthesized quickly, slowly, or not at all. This controls the number of different proteins being made in a cell
- Defines cell and its functions, makes cells alive because it governs cells
- Doesn't contain replication, only transcription and translation
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Gene Expression
- The two main steps in gene expression are transcription & translation
Transcription: mRNA is synthesized based on a DNA template
- Translation: Ribosome assembles amino acids based on the mRNA codons to synthesize a protein
- Gene expression refers to the synthesis of protein based on the DNA sequence of a gene
- Gene expression in eukaryotes involves transcription to form mRNA, which undergoes processing before being exported to the cytoplasm. Once in the cytoplasm, the mRNA is used as the template for protein synthesis by the ribosomes.
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What structural characteristic of A-T bonds make it a good starting point?
- Only 2 hydrogen bonds hold adenine and thymine together, making it an easier bond to break.
- This allows the DNA to unwind easier than at a G-C bond to initiate transcription
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Binding to the promoter region
- Every exon (coding information, exon) starts with a promoter and ends with a terminator
- When RNA polymerase comes into the DNA, it looks for the promoter site b/c of the TATA box
- A promoter region is where the RNA polymerase complex binds to initiate transcription
It is rich in A-T bonds
- example: the TATA box in E. coli (shown above) contains 2 promoter regions
- The promoter is upstream (5’) of the start site. This allows it to bind to the correct strand
- : the TATA box is an EXAMPLE from E. coli, it is not present in every organism, although it shows fairly high conservation across species, with small variations.
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POSTTRANSCRIPTIONAL MODIFICATIONS
- We need to modify our pre-mRNA molecule to allow it to get out of the nucleus and be translated into a polypeptide of amino acids!
- Prokaryotes: mRNA is used immediately
In eukaryotic cells, the primary transcript needs to undergo capping, tailing and base excision before it can leave the nucleus
- Modifications convert precursor mRNA (pre-mRNA) to mature mRNA
Increase stability and resistance in the open cell environment, once outside the nucleus
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Transcription
Transcription of a DNA template produces an RNA molecule that is a copy of the genetic information. The nucleotide sequence of this newly formed mRNA molecule is then translated using the genetic code to produce the protein coded for by the gene.
The entire mRNA transcript consists of two regions:
1. coding region called exons
2. non-coding region called introns
- A coding region refers to a sequence of DNA bases that will produce a protein
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CAPPING
- Cap is added to the 5’ end of mRNA strand (7-methyl guanosine, a modified guanine nucleotide triphosphate)
- Capping has two functions: IMPORTANT
1. protect RNA from digestive nucleases as it exits the nucleus and enters cytoplasm
2. recognized by ribosome to translation
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TAILING
- ~200 adenine ribonucleotides, known as a poly-A tail, is added to the 3′ end of the transcript by an enzyme called poly-A polymerase
- Increases stability, prevents degradation of mRNA near the ends
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BASE EXCISION (Splicing)
- SnRNA's attach on both sides of intron and then gravitate towards each other, pulling the exons towards each other. This forms a splicosome, which has 4 subunits (2 SnRNA and 2SnRNP). This cuts the introns out. RNA ligase glues exons together
- Introns are removed, or else they would prevent translation
- Spliceosomes remove introns (cut them out) and join the exons together so the transcript is one continuous coding gene
- Introns stay inside the nucleus and are degraded into recycled nucleotides
- The “primed” mRNA transcript then moves out of the nucleus and into the cytoplasm where it will be translated (to come…)
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Silent mutation
- Mutation that has no effect on amino acid sequence of a protein
- Can be a change of 1 or more base pairs
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Mutagens
Cause mutations
Physical Mutagens
- They physically change the structure of DNA and range from point mutations to loss of large portions of chromosomes
- get into nucleus and change structure of DNA, more prominant than chemical mutagens
- e.g., X rays, UV Rays, Gamma Rays
Chemical Mutagens
- Can enter the nucleus of a cell and induce mutations by reacting chemically with the DNA. They can cause nucleotide substitutions or a frameshift mutation
- e.g., nitrites, gasoline fumes, cigarette smoke, and any other carcinogens (cancer causing substance).
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TRANSLATION
- Synthesis of a protein from an mRNA template
- Process involves several key molecules:
mRNA
- the small and large subunits of the ribosome
- tRNA and the release factor
- Process is broken into three stages: initiation, elongation, and termination
Initiation of Translation
- Small ribosomal subunits bind to the 5′ cap of the mRNA transcript and translation commences
- mRNA codon AUG-codes for methionine amino acid (START)
- tRNA with anticodon UAC, carrying methionine will bind AUG site on mRNA
- Large ribosomal subunit binds the small subunit now, and activates the complex
Elongation of translation
- Ribosome moves along mRNA from 5’ to 3’, reading the code in triplet codons
When the start codon is in the P site, tRNA has delivered methionine
- Second codon is now in the A site
- Appropriate tRNA delivers the next amino acid in the protein sequence
- Peptide bond is formed between methionine and the second amino acid in the P site
- Then, the Ribosome subunits shift down one codon (moving in 5′ to 3′ direction of mRNA strand)
- mRNA is read 5’-3’
- The polypeptide chain grows while it is in the P site
- The start codon establishes the reading frame – all codons read by the ribosome to form the protein
- Methionine is transferred to the A-site amino acid, the first tRNA exits, the ribosome moves one codon (3 base pairs) along mRNA
- First tRNA goes to pick up another amino acid (met)
- This puts the amino acid chain in the P-site, and frees up the A site for the next tRNA
- As elongation continues, the growing peptide is continually transferred to the A-site tRNA, the ribosome moves along the mRNA, new tRNAs enter, and peptide bonds are formed
- Process of elongation continues until a stop codon is read in the A site
- Stop codons are UAG, UGA and UAA
Termination of translation
- Elongation ends when a stop codon is encountered in the A-site
- A release factor enters the A-site and causes the ribosome subunits to disassemble (small and large subunits fall apart) and translation is terminated, releasing the mRNA and newly formed protein
- Protein is folded and modified and then targeted to areas of the cell where it is required
- mRNA can be retranslated, or can be degraded immediately
- The anticodon is also in RNA format
- PROTEIN SEQUENCE IS STILL READ FROM MRNA MOLECULE
Review
Initiation
- Ribosome binds to a specific site on the mRNA
Elongation
- Ribosome moves along the mRNA 3 nucleotides at a time (5’ – 3’ direction)
- Each set of three nucleotides (called a codon) codes for one amino acid
- tRNA delivers the appropriate amino acid to the mRNA during translation
Termination
- The ribosome falls off the mRNA when it comes across a stop codon
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Transfer RNA (tRNA)
- specific to mature mRNA. To find it, get rid of introns so that you only have mature mRNA, and then find the complimentary to it. Start with UAC (anticodon of start codon, AUG)
- Single stranded RNA folded into a clover shape
- Carry Amino Acids to the ribosome
- The tRNA molecule is a cloverleaf in 2-dimensional shapes, and a ‘boot’ shape in 3 dimensions
- Hydrogen bonds and base pairings twist the clover shape into the boot shape
- There are only 20 amino acids but 61 tRNAs b/c of redundancy, which carries the same amino acids with different codons
Anticodon and Acceptor stem on tRNA
- On the bottom stem loop
- The 3 base portion of tRNA that binds to mRNA
- It is complimentary to the mRNA strand
**Written 3’-5’, since mRNA is 5’-3’
- At the opposite 3’ end of the tRNA is the acceptor stem
- This is where amino acids are attached and carried to the ribosome
- The scanner tries to find its complimentary base pairs, when it does, it attaches to amino acid that is on it. This is why it has anticodon If codon, it wouldn't be able to find its complimentary base pairs
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Polypeptide chain
- A protein is built one amino acid at a time
- Amino acids are added to the chain one by one by the ribosome
- Forms a peptide bond between each amino acid
- Amino acids are carried to the ribosome by tRNA
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Chromosomal Mutations
- A change to the number of chromosomes is always negative, and often lethal
- Eg. trisomy 21 = Down’s Syndrome
- There can also be mutations of entire chromosome sections, which would affect several/many genes (Deletion, Duplication, Inversion, Translocation)
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The Structure of Ribosomes
- Ribosome exists as 2 subunits
- large and small subunits
- When both attach together, they create a ribosome
- The small subunit binds mRNA
- Large subunit binds 3 tRNA molecules at a time
- Begins to grow amino acid chain (polypeptide)
- Each subunit is made of different proteins and rRNA
P-A-E sites of Large ribosomal subunit
- The P (peptide) site holds the tRNA of the amino acid being added to the polypeptide chain
- Forms the peptide bond to link each amino acid
- The A (amino) site accepts the next tRNA holding the next amino acid molecule
- The E (exit) site removes tRNA molecules that have given their amino acids to the chain
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DNA MUTATIONS
- CHANGES IN THE GENETIC MATERIAL OF AN ORGANISM
TWO CATEGORIES:
- Single-Gene Mutations involve changes in the nucleotide sequence of one gene
- Chromosome Mutations involve changes in chromosomes, and may involve many genes
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MISSENSE MUTATION
- Mutation that changes the amino acid sequence of a protein
- This can be good or bad
- It may introduce a new protein that will help an organism survive it’s environment
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polypeptide bonds
- The bond forms between the amino terminal of the first amino acid and the carboxyl terminal of the next amino acid
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POINT MUTATION
- Single-gene mutation resulting from a change in a single base pair
- happen at a specific area in the genetic code, happen within the nucleotides
- Can involve substitution/insertion/deletion of a single base pair
- Substitutions have a fairly minor effect on cell due to redundancy of genetic code
- Eg. A mutation of GGA (glycine) to GGG (glycine), will have no effect on the organism
FRAMESHIFT MUTATION
- Insertion or deletion of nucleotides
- Causes the entire reading frame of gene to be altered (yikes!)
- Causes changes to neighbouring triplets as well
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Proteins
- Once produced, the amino acids sequence (primary protein structure) begins to fold to form a functional 3D protein
- The proteins formed by transcription and translation are sent to the Golgi body for further modification
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A mutation is caused to the DNA of a skin cell. Will this mutation be passed on to future offspring?
Somatic cells vs Gametes. No. Negative mutations that do not provide a selective advantage are typically not passed on. There are DNA repair mechanisms that will repair this mutation before it gets passed along. Even positive mutations (provide an advantage) may be repaired before being passed on.
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Regulation of Gene Expression
- Most genes are not constantly producing proteins – this would require too much energy
- Genes are “turned on” (transcribed and translated) and “turned off” throughout the life of an organism at appropriate times
- Genes that are constantly expressed are called constitutive genes aka “housekeeping genes”
These genes express proteins important to the survival of the organism
- Most genes are regulated, so that the protein is expressed only in certain amounts at certain times when its needed
- Gene regulation involves turning on or off specific genes depending on the needs of the organism
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NONSENSE MUTATION
Mutation that shortens a protein by introducing a stop codon
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Prokaryotic Gene Regulation Controlled at 4 Levels
- Transcriptional - regulates which genes in DNA are transcribed
- Posttranscriptional – mRNA undergoes changes in the nucleus
- Translational – controls how often and how rapidly mRNA transcripts will be translated into proteins
- Posttranslational– control the rate at which a protein becomes active
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lac Operon in prokaryotic E. coli
- positive feedback loop because it's not inhibited and is always off but on whenever needed
- An example of an inducible system to regulate gene expression
- lac Operon consists of a cluster of three genes that code for proteins involved in the metabolism of lactose: The three genes are lacZ, lacY and lacA
- The lac operon contains three genes (Z, Y, and A) needed for the breakdown of lactose in E. coli. All three genes are under the control of one promoter. Therefore, they undergo the same level of regulation. When lactose is not present, the lac repressor inhibits transcription.
- CAP = catabolite activator protein (protein that binds to help produce proteins to catabolize, or breakdown, lactose)
- used a lot in research b/c simple, not very big, not complex, grows quickly, this helps to manipulate DNA easier
Lac Operon “OFF”
- When lactose is absent this inhibits lac protein expression. A repressor protein (Lac1 protein) binds to the operator site.
- this blocks RNA polymerase from binding to the promoter
- no β-galactosidase is produced
- Negative feedback loop. In conditions of low lactose, there is increased activity of the repressor protein, which is decreasing the output of this operon.
Lac Operon “ON”
- The presence of lactose acts as an inducer. A derivative of lactose, allolactose binds to the Lac1 protein (repressor) so it can no longer bind to the operator of the lac operon DNA
- RNA polymerase can now bind to the promoter region and transcribe the lacZ, lacY, lacA enzymes
CAP is an activator
- When the cell detects low glucose, it will attempt to increase lactose metabolism and will therefore need these lactose metabolizing enzymes in high amounts.
- In conditions of low glucose, there tends to be high cAMP which binds to the CAP protein and allows it to attach to the CAP site and greatly increase the binding of RNA Polymerase to the promotor
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Repairing Mutations
- Photorepair – A specific repair mechanism that repairs damage to DNA caused by exposure to UV radiation. A photolyase enzyme recognizes the damage, binds to the site and corrects it
- Excision repair – Non-specific repair mechanism whereby parts of damaged DNA are removed and DNA polymerase puts down new/correct nucleotides
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OPERON’s
- A cluster of genes in a DNA strand under the control of one promoter (only in prokaryotes)
- acts as simple regulatory feedback loop
- consists of a promoter, operator, and two or more protein-coding genes
- Promoters and operators do not code for proteins, but serve as binding sites for regulatory proteins
- When a repressor protein binds to the operator, transcription of the structural genes is inhibited (control)
- Two examples of operons are the lac Operon and the trp Operon
- Inhibition and activation are the ways in which genes are regulated and controlled
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Lactose
- lactose is a disaccharide found in milk/milk products, consists of two sugars (glucose and galactose)
- β-galactosidase is the enzyme responsible for the degradation of lactose
- It is produced by the lac operon in E. coli
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trp Operon of prokaryotic bacteria E. coli
- negative feedback loop because it's inhibited and is always on but off if needed
- Trp Operon in E. coli contains five genes that are involved in the synthesis of essential amino acid tryptophan.
- This operon is normally transcribed, until the cell has sufficient tryptophan. Once enough tryptophan is present for normal cell functioning, the extra tryptophan binds to the repressor protein allowing it to attach to the operator and inhibit transcription.
The trp operon is “OFF”
- Regulates genes for tryptophan (an amino acid) production in prokaryotes
- The trp operon is inhibited when high levels of tryptophan are present
- Tryptophan is a co-repressor because it binds with the trp repressor protein and activates this repressor protein (transcription proceeds)
- This complex will the deactivate (turn off) gene expression of the trp operon
The trp Operon is “ON”
- Lack of tryptophan deactivates the repressor and activates transcription via RNA Polymerase
- RNA polymerase transcribes trp operon genes
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Use of operons in Research
- Both operons are very well studied and understood
- Researchers will insert this gene into bacteria along with a gene being researched
- To confirm that the genes have been inserted, scientists use a modified form of lactose.
- If the gene has been inserted properly, lactose will be digested and turn the bacteria BLUE
- This strain can now be used for research of the other gene attached
- easy to manipulate and is mainly found in prokaryotic cells
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Regulation of Gene Expression in Eukaryotes
- Regulation of eukaryotic gene expression occurs at multiple stages of protein production. The regulation points occur in both the nucleus and the cytoplasm.
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5 levels of regulation
- Pre-transcriptional (DNA)
- Transcriptional (DNA to mRNA)
- Post-transcriptional (mRNA)
- Translational (mRNA to protein)
- Post-translational (protein)
*Note: genes are not organized into operons in eukaryotes.
- Each gene has its own promoter
- Distinct transcription factors control each gene
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Pre-transcriptional and Transcriptional Control
- every gene has its own polymerase which has its own promoter
- DNA level control
- Pre-transcription: Highly condensed chromatin acts as a physical barrier to prevent transcription and the formation of pre-mRNA
- Transcriptional Control: Certain types of activator proteins also enhance transcription initiation by binding to transcription factors and RNA polymerase
- These are known as enhancers
- Having multiple activator regions on a gene allows a gene to be highly tuned to specific environmental factors (like fine tuning your speed while on cruise control in a car)
Transcriptional Control – DNA Level Regulation through Activators and Transcription Factors
Transcription factor binding is required for transcription.
Activators enhance rate of transcription
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Post-Translational
- When the protein comes off the ribosomes, they get tagged and the proteas get signaled by ubiquitin to eat it. This only happens if protein isn't needed.
- factors can attach to protein to stop it from folding
- The two top things are not ideal bc it's a waste
- if needed, other proteins will come and attach to that protein and make it superfold so that it's functional
- Polypeptide level control
- Proteins must be activated by modifications to the amino acid chains
- E.g. Insulin is initially folded into 3D structure
- Removal of amino acids activates it
- Cells can tag a protein for degradation
- Adding a chain of ubiquitin molecules to a protein can signal for protein degradation
- Proteasome complexes notice the ubiquitin tag and will degrade the protein
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Post-transcriptional and Translational Control
- mRNA level control
- Alternative splicing can produce different mRNA molecules
- 5’ Cap and 3’ poly-A tail are purposefully not added (what will happen?)
- RNA interference – the regulation of gene expression by small RNA’s (sRNA); it inhibits gene expression by degrading mRNA or inhibiting translation by binding to it, inhibits the mature RNA from making protein
- Small RNA’s (micro RNA) and small interfering RNA can inhibit translation and interact with specific mRNA’s
- if ribosomal subunits attach, where methionine attaches to p site, translation can be blocked by a site. If a site is blocked, only methionine is in, but won't be enough to fold so it won't be functional
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Recombinant DNA
- DNA made in a lab that consists of material from more than one source
- Different sources are often different species
- Became possible with the discovery of Restriction Enzymes
- Used by bacteria to cut up viral infecting DNA and protect their own genomes from the viral DNA
- Restriction Endonuclease: a unique restriction enzyme that cuts DNA within the strand.
- Does so by recognizing a short sequence (Target sequence) and cleaving it at the restriction site. Different endonucleases recognize different target sequences.
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Restriction Endonuclease Enzyme
- Cut DNA at specific sites
- leave “sticky ends” leftover hanging bits
- palindrome (reads the same in the 5’ to 3’ direction)
- Cuts are made at the restriction sites
EcoRI
- Each restriction enzyme that we discover is known to recognize a specific sequence
- EcoRI is a restriction endonuclease that will recognize this DNA sequence (notice that it is a palindrome
- Recognition sequence always the same 5’ to 3’
- Restriction fragments with sticky ends form base pairs w/ other single stranded regions with the correct complimentary sequence
- This is very specific, as all the nucleotides must match to bond
- Blunt cuts reduce specificity
- No sticky ends that can form complimentary base pairs
- Two fragments with blunt end can combine
- Less efficient
- Only 2 nucleotides must match, higher chance that unwanted DNA will bind to this site
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How to Make Recombinant DNA (mixed DNA)
1. A restriction endonuclease is selected
Can cut both of the DNA fragments that
- we want combined
2. Each DNA piece is reacted with the restriction endonuclease to produce cut fragments that will have matching sticky ends
3. Cut DNA fragments are incubated with DNA ligase (joins strands)
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Why would we want to be cutting DNA and making mixed recombinant DNA?
- Creating recombinant DNA in a lab allows us to express proteins in larger quantities than they normally would be in vivo (occurring naturally in a living thing)
- Expression of our new recombinant DNA can be controlled when we pair our recombinant DNA with an inducible gene (lac operon)
- Straight forward process, carried out in microlitre quantities
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Gene Cloning in Bacteria
- Gene Cloning: making multiple identical copies of a gene or segment of DNA using bacteria
- Transformation: bacterial DNA takes up the inserted DNA under specific conditions
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How to Clone Genes in Bacteria
1. In bacteria, the vector (carrier of gene) is a plasmid (circular DNA)
- Contains an origin of replication (plasmid will be replicated and included in the daughter cells)
- Plasmids are grown to have resistance to the antibiotic Ampicillin.
- Plasmid has a restriction endonuclease site within the lacZ gene… if the new DNA is added here, it will deactivate the lacZ gene
- Selectable markers allow researchers to identify which bacteria are the bacteria that contain the recombinant DNA.
2. Transformation
- process for which foreign DNA is taken up by bacteria
- Certain chemicals make cell membrane porous, allows it to take in DNA
3. Bacteria cells grow in a Petri dish
- Growth Gel in the petri dish contains antibiotic Ampicillin and
X-gal to makes bacteria blue when broken down by the enzyme from the lacZ gene
- If the cells turn blue, we know that the lacZ gene is active and our recombinant DNA was not added successfully
- Selectivity is arguably the most important step. We need to be able to identify which colonies have this gene in them
4. Bacteria with recombinant DNA are ID’d thanks to the selective markers
- Bacteria growing contains recombinant DNA or plasmid
- Blue bacteria contains an active lacZ gene to therefore we know that no gene was inserted in these bacteria
- White colonies contain recombinant DNA
- *Selectivity*: We will know if the bacteria has the inserted DNA if:
1) it grows (possesses antibiotic resistance gene)
2) it stays white
- if it turns blue, the lacZ gene is active
- if it is active, it was not cleaved by the R.E
- if it was not cleaved, the DNA could not have been inserted
5. Cells with recombinant DNA are selected and grown in liquid culture
6. Recombinant DNA molecules are isolated and purified from bacterial cells
7. Analysis techniques are used to confirm correct recombinant DNA is made
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Polymerase Chain Reaction (PCR)
- DNA Amplification: production of large quantities of DNA from a sample
- Polymerase Chain Reaction (PCR): Automated method for amplifying specific regions of DNA from very small quantities
- Produces billions of copies of a section of DNA in a test tube within hours
- Very important in Forensics = amplify a very very small amount of a suspects DNA into a quantity that is testable for distinguishing markers
The Polymerase Chain Reaction
1. Double strand DNA heated to 94-96⁰C to denature it into single strands (split it up)
2. Cooled to 50-60⁰C to allow RNA primers to anneal(bind) to the strands
3. Heated to 72⁰C, where a special heat-resistant DNA polymerase (Taq Polymerase) can optimally function and add complimentary nucleotides from the primers
4. Cycle repeats 30 cycles to produce 1 billion copies of initial strand
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Analyzing DNA
- Once DNA has been amplified, there are various methods for analyzing and identifying it
- One standard method is Gel Electrophoresis: used to separate DNA molecules according to mass/charge
- DNA is negative due to its phosphate (negative charges will repel it)
- DNA can be cut into different sizes (larger fragments move slower in gels)
Made up of:
- Agarose, a protein gel that allows DNA to separate and move within it
- Electrodes (positive/negative electric charges)
- Separate wells to add samples
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Gel Electrophoresis Procedure
1. Gel is treated with ethidium bromide and added to wells
- The dye binds the backbone of DNA and fluoresces(glows) under UV light
- Helps us to see the DNA on the gel
2. Gel is placed in buffer and electric current is run from cathode(-) to anode(+)
- Negatively charged DNA (due to phosphate groups) will move from the negative end towards the positive end
- Buffer maintains pH of solution (keeps DNA from denaturing)
3. Smaller DNA fragments move faster/further than larger fragments
- They fit through the agarose gel easier
4. Gel is removed from buffer and exposed to UV light
- Ethidium bromide in the DNA backbone glows and allows us to see the DNA
- A ladder is a lane that contains DNA of known size (base pair length)
- We compare the fragments in the gel to this standard
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DNA Fingerprinting for ID
- a.k.a DNA profiling
- Each individual has unique DNA (except identical twins)
- In the past Restriction Fragment Length Polymorphism (RFLP) was used
Cut up DNA and run on a gel
- Current method is short tandem repeat (STR) profiling
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5' to 3' ends
- the 5' carbon is on the fifth end, which has a phosphate group
- the 3' end has an exposed OH group on the 3' end of the carbon
- DNA is always built 5'-3' because the exposed OH on the 3' can be removed to add more nucleotides there (condensation reaction)
- replication makes DNA that has two strands that are anti-parallel, this is because this maximizes hydrogen bonding b/c the two nitrogenous bases are closer together, making it easier to have hydrogen bonds
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Amino acids
There are 20 amino acids
- 8 are essential (taken in by food)
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codons
- with the start codon (AUG), we don't produce it, but its the start to every single protein
- The reason why some codons are repetitive and end up being the same name is because it's a failsafe incase of mutations
- Runs from N to C
