how many processes can you name?
DESCRIPTION
Lecture 0 Basics of Molecular Biology Welcome to lecture 0! SynBUM | MIT iGEM Team 2010 Create Your Own Bacterial Air Freshener 1/6/2011TRANSCRIPT
How many processes can you name?
The Big Picture (Shown at very beginning of class, before everyone
is seated) If you would like to see a narrated version, its here:
How many processes can you name? Lecture 0 Basics of Molecular
Biology
Welcome to lecture 0! SynBUM | MIT iGEM Team 2010 Create Your Own
Bacterial Air Freshener 1/6/2011 What you will learn in this
lecture
The cell as the basic unit of life Structure of Important
Macromolecules DNA RNA Proteins The Central Dogma Transcription
Translation Regulation Correlation to Synthetic Biology Cells -
Fundamental working units of every living system.
Organelles Nucleus (contains DNA) Cytoplasm Membrane DNA (no
nucleus) Eukaryotic cell Prokaryotic cell Every organism is
composed of one of two types of cells: prokaryotic cells or
eukaryotic cells. A eukaryotic cell has membrane-enclosed
organelles, the largest of which is usually the nucleus By
comparison, a prokaryotic cell is simpler and usually smaller, and
does not contain a nucleus or other membrane-enclosed organelles
Bacteria and Archaea are prokaryotic; plants, animals, fungi, and
all other forms of life are eukaryotic Prokaryotes andEukaryotes
are descended from the same primitive cell all life on Earth is the
result of 3.5 billion years of evolution. The cell is the lowest
level of organization that can perform all activities required for
life All cells: Are enclosed by a membrane Use DNA as their genetic
information The ability of cells to divide is the basis of all
reproduction, growth, and repair of multicellular organisms
Comparison of Prokaryotic and Eukaryotic Cells
Prokaryotes Eukaryotes Single cell Single or multi cell E. coli
chromosome: 4X106 bp Yeast chromosome: 1.35x107 bp 90% of DNA
encode protein Small fraction of DNA encodes protein: Many repeats
of non-coding sequences No nucleus Nucleus No organelles Organelles
One piece of circular DNA Chromosomes No mRNA post transcriptional
modification Exons/Introns splicing Prokaryotic cells are generally
chosen for genetic engineering because: Processes are better
understood to engineer something intelligently we need to
understand what were changing first! Easier to maintain, less
specialized equipment needed Faster, easier to scale up However, as
can be seen in the last iGEM project (well talk about this later),
Eukaryotic cells also have a wide range of outputs. In our class
well be using prokaryotic cells All Cells Divide All cells go
through similar cycles: they eat, grow, replicate their DNA,
divide, and repeat The microraphs have the cytoskeleton stained,
showing cell division: Above: early anaphase Below: telophase Cell
Cycle: The Chromosomal View
(b/c its pretty ) The Central Dogma DNA RNA Proteins
Control/ Info Center -Genes -Regulatory elements The messenger
-mRNA, tRNA, rRNA Also can be: -Ribozymes -siRNA The machinery
-Enzymes -Signaling -Replication -Many more DNA Replication The
central dogma is just what it sounds like: it is central to our
current understanding of biology. Information encoded in DNA is
passed to RNA and expressed as proteins. Control/Info Center: (DNA
& RNA): Stored as nucleic acids: biological molecules(DNA and
RNA) Specific sequences of DNA bases that encode instructions on
how to make proteins. Machinery/Factory + Products (Proteins):
Building blocks of cells Collect and manufacture components, form
enzymes Carry out replication Genotype: The genetic makeup of an
organism (whats inside) Phenotype: the physical expressed traits of
an organism (what you see) Of course, in recent years weve found
that the cell has many levels of regulation beyond the simple
DNA->RNA->Protein path. However, for this lecture we will
focus on the central dogma. DNA Transcription RNA Translation
Proteins Information encoded in DNA is passed to mRNA Information
carried by mRNA is used to make proteins But first, what are DNA,
RNA, and Proteins? Where are we? The cell as the basic unit of
life
Structure of Important Macromolecules DNA RNA Proteins The Central
Dogma Transcription Translation Regulation Correlation to Synthetic
Biology DNA: The Code of Life 5' end 5'C 3'C Nucleoside Nitrogenous
base
(b) Nucleotide Nucleoside Nitrogenous base Phosphate group Sugar
(pentose) DNA: Deoxyribonucleic acid Polymer nucleotides Purines
(these are bigger): Adenine and Guanine Pyrimidiens (Smaller):
Thymine and Cytosine The two strands are held together by H-bonds A
pairs with T, C pairs with G Antiparallelhas a 3 (phosphate) end
and a 5 (hydroxyl) end DNA encodes many things: Genes that tell the
cell how to make proteins As well as tandem repeats, trash,
regulatory elements, protein binding sites, etc Theres still a lot
we dont know about DNA! Nucleoside triphosphate
DNA Replication A C T G New strand 5 end Template strand3 end 5 end
3 end Nucleoside triphosphate Pyrophosphate DNA polymerase
Semiconservative Template DNA is used by DNA polymerase to
synthesize the new strand Proceeds only from the 5 to 3 end (Aside)
For the chemists, why this this happen? Answer: polymerization
requires a free hydroxyl group DNA Replication (E. Coli)
Leading strand Lagging strand Origin of replication Primer Overall
directionsof replication Origin of replication Template Strand New
Strand dsDNA (a) Origins of replication in E. coli 0.5 m DNA
Replication (Eukaryote)
0.25 m Origin of replication Double-stranded DNA molecule Template
Strand New Strand Bubble Replication fork Two daughter DNA
molecules (b) Origins of replication in eukaryotes RNA RNA DNA
Single Stranded Double Stranded Temporary (mRNA) Stable
Uracil Thymine Ribose Deoxyribose mRNA tRNA rRNA miRNA/siRNA
Ribozymes Main differences between RNA and DNA. There are many
types of RNA known Shown are two view of tRNA tRNA linear and 3D
view: Main types of RNA mRNA this is what is usually being referred
to when we say RNA.This is used to carry a genes message after
transcription tRNA transfers genetic information from mRNA to an
amino acid sequence during translation rRNA ribosomal RNA.Part of
the ribosome which is involved in translation Proteins are made of
Amino Acids
group Carboxyl carbon Proteins are a chain of 20 different amino
acids different chemical properties cause the protein chains to
fold up into specific three-dimensional structures that define
their particular functions in the cell. 3 letters of RNA(/DNA),
called a codon 1 amino acid 64 possible combinations map to 20
amino acids Degeneracy of the genetic code- several codons to same
amino acid Proteins do all essential work for the cell build
cellular structures digest nutrients execute metabolic functions
Mediate information flow within a cell and among cellular
communities. Proteins work together with other proteins or nucleic
acids as"molecular machines" structures that fit together and
function in highly specific, lock-and-key ways. Proteins are the
Workhorses of the Cell
Primary Structure Secondary Structure Tertiary Structure Quaternary
Structure pleated sheet +H3N Amino end Examples of amino acid
subunits helix A ribbon model of lysozyme (a) (b) A space-filling
model of lysozyme Groove Primary structure: amino acid sequence,
determined by DNA Secondary structure: Hbonding between backbone
Tertiary structure: Hbonding, Hydrophobic forces, disulfide bonds,
etc. between R groups Quaternary structure: how many polypeptides
fit together The shape of a protein is extremely important for its
function! Little changes in shape can lead to nonfunctional
proteins. Where are we? The cell as the basic unit of life
Structure of Important Macromolecules DNA RNA Proteins The Central
Dogma Transcription Translation Regulation Correlation to Synthetic
Biology The Central Dogma Revisited
DNA molecule Gene 1 Gene 2 Gene 3 template strand TRANSCRIPTION
TRANSLATION mRNA Protein Codon Amino acid Slight Variations of the
Central Dogma
(b) Eukaryotic cell TRANSCRIPTION Nuclear envelope DNA Pre-mRNA RNA
PROCESSING mRNA TRANSLATION Ribosome Polypeptide TRANSCRIPTION DNA
mRNA TRANSLATION Ribosome Polypeptide a) Bacterial Cell
Transcription Terminology
Phosphodiester Bond Promoter RNA (ribonucleotide) RNA Polymerase II
Terminator Phosphodiester Bond: Esterification linkage between a
phosphate group and two alcohol groups. Promoter: A special
sequenceof nucleotides indicating the starting point for RNA
synthesis. RNA (ribonucleotide): Nucleotides A,U,G, and C with
ribose RNA Polymerase: Multi-subunit enzyme that catalyzes the
synthesis of an RNA molecule on a DNA template from nucleoside
tri-phosphate precursors. Terminator: Signal in DNA that halts
transcription. Transcription Initiation Elongation
Termination
Promoter Transcription unit DNA Start point RNA polymerase 5 3
Initiation 1 RNA transcript Unwound Template strand of DNA 2
Elongation Rewound 3 Termination Completed RNA transcript
Transcription 3 Main stages: Initiation, elongation, and
termination Catalyzed by RNA Polymerase Eukaryotes process mRNA;
this does not occur in prokaryotes. Transcription occurs in the
nucleus. Initiation Promoters signal the initiation of RNA
synthesis Transcription factors mediate the binding of RNA
polymerase and the initiation of transcription The completed
assembly of transcription factors and RNA polymerase II bound to a
promoter is called a transcription initiation complex A promoter
called a TATA box is crucial in forming the initiation complex in
eukaryotes Elongation As RNA polymerase moves along the DNA, it
untwists the double helix, 10 to 20 bases at a time Transcription
progresses at a rate of 40 nucleotides per second in eukaryotes A
gene can be transcribed simultaneously by several RNA polymerases
RNA polymerase II catalyzes the formation of phosphodiester bond
that link nucleotides together to form a linear chain from 5 to 3
by unwinding the helix just ahead of the active site for
polymerization of complementary base pairs. The hydrolysis of high
energy bonds of the substrates (nucleoside triphosphates ATP, CTP,
GTP, and UTP) provides energy to drive the reaction. During
transcription, the DNA helix reforms as RNA forms. Termination The
mechanisms of termination are different in bacteria and eukaryotes
In bacteria, the polymerase stops transcription at the end of the
terminator In eukaryotes, the polymerase continues transcription
after the pre-mRNA is cleaved from the growing RNA chain; the
polymerase eventually falls off the DNA Translation
Terminology
Polypeptide Ribosome Amino acids tRNA with amino acid attached tRNA
Anticodon Trp Phe Gly Codons 3 5 mRNA Translation Terminology Codon
mRNA Ribosome rRNA tRNA Anti-codon C-Terminal N-terminal Codon: The
sequence of 3 nucleotides in DNA/RNA that encodes for a specific
amino acid. mRNA (messenger RNA): A ribonucleic acid whose sequence
is complementary to that of a protein-coding gene in DNA. Ribosome:
The organelle that synthesizes polypeptides under the direction of
mRNA rRNA (ribosomal RNA): The RNA molecules that constitute the
bulk of the ribosome and provides structural scaffolding for the
ribosome and catalyzes peptide bond formation. tRNA (transfer RNA):
The small L-shaped RNAs that deliver specific amino acids to
ribosomes according to the sequence of a bound mRNA. Anticodon: The
sequence of 3 nucleotides in tRNA that recognizes an mRNA codon
through complementary base pairing. C-terminal: The end of the
protein with the free COOH. N-terminal: The end of the protein with
the free NH3. Translation Accuracy Accurate translation requires
two steps:
P site (Peptidyl-tRNA binding site) A site (Aminoacyl- tRNA binding
site) E site (Exit site) mRNA binding site Large subunit Small Next
amino acid to be added to polypeptide chain Amino end Growing
polypeptide tRNA E P A Codons 5 3 Requires 2 correct matches:
Between tRNA and correct amino acid Between tRNA codon and mRNA
anticodon Accurate translation requires two steps: First: a correct
match between a tRNA and an amino acid, done by the enzyme
aminoacyl-tRNA synthetase Second: a correct match between the tRNA
anticodon and an mRNA codon Flexible pairing at the third base of a
codon is called wobble and allows some tRNAs to bind to more than
one codon Ribosomes facilitate specific coupling of tRNA anticodons
with mRNA codons in protein synthesis The two ribosomal subunits
(large and small) are made of proteins and ribosomal RNA (rRNA)
Building a Polypeptide
The three stages of translation: Initiation Elongation Termination
All three stages require protein factors that aid in the
translation process RNA to Protein: Instruction Book of Life
Start with Methionine End with a stop codon Note the degeneracies
for each amino acid Translational Initiation
Large ribosomal subunit 3 U 5 A C P site Met 5 A Met U G 3
Initiator tRNA GTP GDP E A mRNA 5 5 3 3 Start codon The initiation
stage of translation brings together mRNA, a tRNA with the first
amino acid, and the two ribosomal subunits First, a small ribosomal
subunit binds with mRNA and a special initiator tRNA Then the small
subunit moves along the mRNA until it reaches the start codon (AUG)
Proteins called initiation factors bring in the large subunit that
completes the translation initiation complex Small ribosomal
subunit mRNA binding site Translation initiation complex
Translational Elongation
Amino end of polypeptide mRNA 5 3 E P site A GTP GDP Ribosome ready
for next aminoacyl tRNA During the elongation stage, amino acids
are added one by one to the preceding amino acid Each addition
involves proteins called elongation factors and occurs in three
steps: codon recognition, peptide bond formation, and translocation
Translational Termination
Release factor 3 5 Stop codon (UAG, UAA, or UGA) 2 Free polypeptide
2 GDP GTP Termination occurs when a stop codon in the mRNA reaches
the A site of the ribosome The A site accepts a protein called a
release factor The release factor causes the addition of a water
molecule instead of an amino acid This reaction releases the
polypeptide, and the translation assembly then comes apart
Almost-Unsimplified Overview
Based on crystallographic data, real protein structures The only
simplifications are: time scale, less crowded background Where are
we? The cell as the basic unit of life
Structure of Important Macromolecules DNA RNA Proteins The Central
Dogma Transcription Translation Regulation Correlation to Synthetic
Biology The cell is not a sac of chemicals
Homeostasis Levels of Gene Regulation Pre-transcriptional
Pre-translational Post-translational Gene/protein interactions
Negative Feedback Positive Feedback A cell is much more than just a
sac of chemicals. It is highly regulated and ordered. The process
of homeostasis maintains a balance between processes and conditions
inside and outside the cell Regulation of a gene can occur at 3
different levels Pre transcriptional: this is the most energy
efficient level. For example, transcription factors stimulate or
prevent transcription of a certain gene. There are also other ways
to regulate the gene, such as chromatin condensation through
histone modifications Pre-translational: this can be: alternate
splicing of a gene Increased degredation/ stability of mRNA mRNA
interference Increase degredation Prevent docking onto ribosome
Post-translational: most energy cost, after protein has already
been made Increased degredation Protein cleavage: can activate
protein Phosphorylation Feedback mechanisms allow biological
processes to self-regulate Negative feedback means that as more of
a product accumulates, the process that creates it slows and less
of the product is produced Positive feedback means that as more of
a product accumulates, the process that creates it speeds up and
more of the product is produced Negative Feedback A Negative
feedback Enzyme 1 B D Enzyme 2 Excess D
blocks a step Negative feedback D C B A Enzyme 1 Enzyme 2 Enzyme 3
Positive Feedback W Enzyme 4 X Positive feedback + Enzyme 5 Excess
Z Y
stimulates a step Z Positive feedback Enzyme 4 Enzyme 5 Enzyme 6 Y
X W + Protein Interactions within the cell
Theres still a lot more we dont know!! Synthetic Biology: How to
get the cell to do what we want?
Mix and match promoters, regulatory sites, and coding sequences to
build logic circuits Does this look familiar to anyone? This is the
circuit the 2010 iGEM team built in bacteria. UV lighttogglephage
polymerization + fluorescence. The possibilities are endless!
Control signaling pathways Search and destroy: cancer cells,
pathogens Control metabolic pathways Clean up oil spills Make novel
biomaterials Hijack the cells differentiation pathways to direct
differentiation T-cell differentiation cure AIDS Make artificial
organs Make new pathways Electricity generating bacteria Can you
think of a few? Visit previous iGEM websites to see what other
college students have done!