Central Dogma and DNA Replication

• The goal is to make a protein.

Central Dogma Theory

• It describes how to make a protein.
• DNA → RNA → Proteins
• Replication (happens in DNA) → Transcription (Happens in RNA) → Translation → Polypeptide
• Polypeptide makes up proteins.
• The flow of information stops at the proteins.

DNA Replication Terms

• Genome is the complete set of DNA in all living things, containing genetic information.
• Genes are specific DNA sequences which code for proteins/make instructions for a specific protein.
• Not every cell needs the same proteins, allowing cells to fulfill their specialized function, known as gene expression.
• Gene expression = transcription + translation

Replication Importance

• DNA Replication is necessary for mitosis (cellular division).
• This allows organisms to grow, develop, and/or repair any tissue; there are millions of strands of DNA in every cell of the body.

What is DNA?

• A double helix strand held together by hydrogen bonds, made up of nitrogen base, phosphate groups, and sugar.
• DNA includes the sugar-phosphate backbone.

DNA Structure

• DNA is made up of nucleotides linked together by phosphodiester (covalent) bonds.
• Phosphodiester bonds are bonds between the phosphate group and a sugar molecule.
• Nucleic acids (DNA & RNA) are always built in a 5' - 3' end.
• Phosphate groups link carbon 5' in one sugar to carbon 3' in another sugar.
• DNA has an anti-parallel structure.
• Nucleotides are always added to the new strand at the 3' end.

How DNA is Built

• DNA is built using Nucleotides.
• DNA always builds from the 5' - 3' end.
• When DNA is linked, base pairs must be added in the following order: A-T and C-G.
• These are paired together using hydrogen bonds.
• Since DNA is anti-parallel, the opposite strand runs in a 5' - 3' direction.

DNA Replication Start

• Prokaryotes (single celled, lacks a nucleus)
• A large protein complex binds to the origin of replication (ori) and starts to unwind the chromosome.
• Eukaryotes (has a nucleus)
• These have multiple origins of replication (ori) which forms replication forks.
• Two replication forks exist in each bubble, going opposite directions.
• Each replication fork is ½ a bubble.

How to Unwind DNA (using proteins)

• DNA Helicase uses energy from ATP hydrolysis to unwind the DNA.
• It works with other proteins, it never works alone.
• It forms replication sites (ORI).
• Single-stranded binding proteins
• Keep the strands from getting back together and bind to single stranded DNA.
• Help to protect the DNA and pair DNA back together.
• Topoisomerase
• It prevents twisting by making small cuts to alleviate pressure in twisted DNA.

When DNA Gets Unraveled

• Two strands are created.
• Continuous (leading) strand
• DNA is replicated from the leading strand, which always has a free 3' end.
• The continuous (leading) strand can synthesize normally following in the 5' -> 3' direction.
• Discontinuous (lagging) strand
• DNA is replicated as the lagging strand, which does not have a free 3' end.
• The discontinuous (lagging) strand cannot synthesize 3' -> 5' and has multiple mini strands formed called okazaki fragments.

Leading Strand

• DNA polymerase 3 is the protein that preforms the function for DNA to be synthesized.
• DNA polymerase is only able to start building DNA with a primer (like a docking site).
• A primer comprises a short RNA starter strand built on the DNA to initiate DNA replication.
• DNA polymerase 3 then adds nucleotides to the 3' end.
• Once DNA is complete, the primer is degraded by DNA polymerase 1 and replaced with DNA.

Lagging Strand

• A new primer synthesized by the primase is needed each time a new okazaki fragment starts.
• Because DNA cannot be built in the 3' -> 5' direction, and if the top template strand is 3' -> 5', then the other template strand is 5' -> 3' due to the antiparallel nature of DNA.
• DNA polymerase 3 adds nucleotides to the 3' end until it reaches the primer of the previous fragment.
• Okazaki Fragments allow for building DNA in the 5' -> 3' direction just like the leading strand.
• DNA polymerase 1 is a protein that replaces the primer with DNA
• Here is how it works:
• It binds and then replaces the primer with DNA
• When replacing the primer, it leaves a gap.
• This gap is closed by DNA ligase which acts like “glue” between gaps. The final phosphodiester linkage between fragments is catalyzed by DNA ligase.

Lecture 14: Central Dogma

• The flow of information stops at the protein.
• Coding DNA (genes) → Transcription → RNA → Translation→ Polypeptide
• Monomer for DNA: nucleotide
• Monomer for RNA: nucleotide
• Monomer for polypeptide: amino acid
• Noncoding DNA (all the rest of the DNA that is not making proteins)
• Noncoding RNA
• Still gets transcribed but does not go to the polypeptide and instead stops at the RNA.
• Two types of noncoding RNA
• rRNA (ribosomal RNA)
• tRNA (transfer RNA)
• Some DNA functions as regulatory sequences
• Technically noncoding but helps regulate the central dogma.
• This mainly occurs at central dogma in eukaryotes
• DNA replication uses DNA as a template.
• It happens in the nucleus.
• Transcription happens in the nucleus.
• DNA never moves out of the nucleus.
• mRNA (M = messenger)
• Carries gene from DNA to RNA.
• Processes the RNA in the nucleus to mRNA.
• Once this occurs, it is ready for replication.
• mRNA gets transferred to the cytosol.
• Translation occurs here.

Transcription

• It is the formation of a specific RNA sequence from a specific DNA sequence.
• Translation is translating nucleotides to amino acids.
• Requirements:
• DNA template
• Monomers
• Nucleotides that will build on the monomers
• Four bases of RNA (AGCU)
• An RNA polymerase
• Enzyme that puts monomers together
• RNA polymerase is what makes RNA during transcription.
• It catalyzes addition of nucleotides to growing mRNA in a 5'-to-3' direction.
• It can only add to the 3' end, so it's “DNA dependent". Needing a DNA template to build off of.
• One RNA polymerase binds the DNA, keeps adding nucleotides, moves along DNA, and adds nucleotides.
• It makes the entire RNA strand and tons of mRNA molecules from one strand.
• Protein opens up the DNA and builds the RNA molecule off of the DNA.
• RNA polymerase catalyzes addition of nucleotides to the 3' end, growing mRNA in the 5'→3' direction.
• The same RNA polymerase binds onto the DNA to add nucleotide; it is not a new polymerase every time.
• Steps involved:
• Initiation
• Elongation
• Termination
• RNA polymerase binds to the promoter, which is a specific point where to transcribe.
• Promoters are DNA sequence before the start of a gene that directs enzymes to where to start and which DNA strand to transcribe.
• The end of the promoter is the initiation site.
• Sigma factors + transcription factors are helper proteins that bind to DNA sequences and to RNA polymerase.
• They direct polymerase onto the promoter and help determine which genes are expressed and when.
• RNA does not stay bound to the DNA.
• The initiation site is where nucleotides are added.

Elongation

• RNA polymerase unwinds DNA and reads 3' → 5' but synthesizes RNA 5' → 3'.
• The transcript is complementary + antiparallel to the DNA template.
• RNA polymerase uses ribonucleoside triphosphates as substrates.
• Enzyme is what is creating those phosph bonds.
• Two phosphate groups are removed from each substrate.
• Energy is released from breaking the phosphates off of the monomer—used to drive polymerization (where monomers link together to form larger polymers).
• The RNA molecule is coming out of RNA polymerase, not bound, closing up the double helix as the polymerase moves about the sequence.
• It synthesizes nucleotides from 5'-3'.

Termination

• Is specified by a specific DNA sequence to stop transcription.
• The RNA molecule and RNA polymerase are released.
• Additional proteins are involved in recognizing termination sites and separating new mRNA, DNA, and RNAP.
• Upstream: toward the 5' end of coding strand or mRNA transcript
• Downstream: toward the 3' end of coding strand or mRNA transcript

RNA Processing (Mechanisms)

• DNA processing does not happen in prokaryotes
• Splicing
• Here introns are removed and exons are joined together, making mRNA smaller than pre-mRNA.
• This is aided by enzyme complexes and occurs In DNA.
• Adding a "cap” and a “tail”
• A 5' cap is added at the 5' end which facilitates mRNA biding to a ribosome and protects mRNA from being broken down/ degraded.
• A poly A tail is added at the 3' end which assists in export from the nucleus and is important for stability of mRNA.
• Poly a tail is important for stability of mRNA
• It helps move RNA from the nucleus to the cytosol.

Lecture 15: Translation

• Goal: genes → proteins
• Genes start in the DNA → transcribed into mRNA → then leaving the nucleus.
• Genetic codes will be used to figure out how we go from nucleotide to amino acid
• Codon: a sequence of three bases that specifies a particular amino acid
• Multiple codons can code for the same amino acid
• Each codon can only specify for ONE amino acid
• Genetic code: specifies which amino acids will be used to build a protein

Map of Translation

• The mRNA transcript is organized in codons.
• It is recognized by tRNA
• What is tRNA
• It binds to specific amino acids and transfers them into ribosomes.
• It has an anti-codon, which is complementary to mRNA codon and matches the codon to the mRNA.
• It carries an amino acid called “charging," which requires ATP, and noncovalently interacts with ribosome and can be recycled.
• This all occurs through interactions with ribosomes.
• Ribosome holds mRNA and tRNA in the correct position to assemble a polypeptide.
• The Translation sequence occurs at 3 sites: E, P, and A

Steps of Translation

• Initiation
• A complex forms around the mRNA
• It has a small ribosomal subunit.
• It is charged (carrying an amino acid) tRNA - The first amino acid in a polypeptide is always methionine (sometimes taken out later).
• There is also a large ribosomal subunit
• All is facilitated by initiation factors, energy input
• Elongation
• After the initial tRNA enters the P site in Initiation, the next codon is “open” for another charged tRNA in the A site.
• The large subunit catalyzes a two-step reaction:
• The bond between the tRNA and its amino acid in the P site is broken.
• A peptide bond forms between that amino acid and the amino acid attached to the tRNA in the A site.
• Therefore, the growing peptide is now in the A Site
• The ribosome shifts down the RNA
• The growing polypeptide is now in the P site instead of the A site.
• The P site is where we want to have the growing polypeptide
• Have two amino acids in P site
• The first tRNA (no longer with its methionine) is now in E site
• tRNA exits from E site
• Now we are ready to add our next amino acid
• The next charged tRNA enters the A site (tRNA anti-codon matches mRNA codon).
• Growing peptide chain transferred to next amino acid
• Charged tRNA in E site dissociates from the ribosome and can be charged again.
• Ribosome shifts
• Old tRNA in E site
• We can do this all all over again until we reach the stop codon.
• Termination
• Stop codon a. in the A site
• Stop codon binds a release factor (a protein)
• The releases causes a dissociation from everything, and it hydrolyzes bond between the peptide and the tRNA in the P site.
• The polypeptide then separates from the ribosome and folds into its 3D shape after possible modification.
• Can make multiple proteins from one RNA

After Translation

• Proteins have specific functions, so it has to go to the correct location.
• Some of the proteins are modified during Post-translational modification.
• Specific Functions: A signal sequence
• The protein indicates where in the cell the new protein belongs.
• Depending on the final location.
• The translation may move to the rough endoplasmic reticulum (RER) proteins bound for organelles in the endomembrane system, or for export from the cell.
• It may also begin in the cytosol. Starting protein synthesis means the ribosome starts translation.
• If there is a signal sequence that indicates it is supposed to be somewhere else, the protein moves to the RER.
• Or translation remains in cytoplasm, so translation occurs there.
• If the protein is destined for the nucleus, the nuclear localization sequence allows entrance to the nucleus.
• Some proteins are modified:
• Phosphorylation
• Addition of phosphate groups catalyzed by protein kinases
• Glycosylation
• Addition of sugars to form glycoproteins
• Adding some carbohydrates
• Proteolysis
• Cutting up protein
• Polypeptide is cut by proteases (e.g., signal sequence is removed)

Lecture 16: Gene Expression and Regulation

• Why do we think about gene expression?
• In multicellular organisms, every cell has the complete genome, but not all genes are expressed in every cell.
• In single celled organisms, certain proteins are made at certain times.
• Only when we need them.
• Gene expression is precisely regulated.
• Important : there are multiple genes on one chromosome. Also this applies to chromosomes, where there are many chromosomes in a genome.

When does gene regulation occur?

• It occurs during and in between transcription and translation
• It is most effective before transcription
• Regulation is used so resources aren't wasted to make RNA that is not needed.
• Types of Gene Regulation
• Inducible genes (control) which are genes that can be turned on, but are normally off.
• Catabolic pathways are inducible since they are usually off until substrates that can be broken down arrive, for example lactose.
• Repressible genes (control) are genes that can be turned off, and are normally on.
• They are used for anabolic pathways, and will turn off when there is enough product because there is to need to keep building any more.

How to Regulate Genes

• Through constitutive genes that are always on and always expressed at a consistent rate, making proteins that are needed by most cells.
• Prokaryotic Gene Regulation.
• This regulates transcription to respond to changes in environment,
• Regulation is also used for needed related functions of sigma factors.
• Many genes share promoters (operons) and therefore can get things done at the same time, including using inducible and repression operons.
• Sigma Factors bind to RNA polymerase and direct the machinery to certain promoters.
• If sigma factors are regulated, then it will also regulate if RNA polymerase can transcribe certain genes.
• Operons are when prokaryotic cells share one promoter that allows multiple genes to share the starting point for RNA polymerase.
• How Operons Work
• They need a promoter, which a DNA sequence where RNAP initially binds.
• They need an operator, a short DNA sequence between the promoter and the structural genes where the binding site for regulatory proteins is.
• Where genes are controlled.
• Two or more structural genes (genes that are used using for proteins)
• Regulatory gene codes code for a protein that regulates gene expression
• A regulatory gene makes a protein that helps regulate if the operon has transcribed or not
• Lac Operon helps E.coli to digest lactose only when it is present and is an inducible operon regulated by a repressor protein that breaks down lactose.

Properties of the LAC Operon

• Has a promoter and operator with three inducible structural genes and a regulatory gene that requires a repressor protein.
• In the lac operon, a repressor protein can bind to the operator, blocking transcription.
• When lactose is absent, the repressor prevents the binding of RNAP to promoter.
• When lactose is present, it will bind to the repressor and change its shape and the repressor leaves the operator, and RNAP can bind to the operon.
• RNAP does not work alone! We use three proteins (ZYA) in the operon to break down lactose in E.Coli which are all inducible.

TRP Operon

• A repressible operon that encodes to synthesize tryptophan (an anabolic pathway) When enough tryptophan is present, the operon is turned off, because if the tryptophan exists the operon is normally turned on, which can be repressed.
• 5 enzymes are needed in the metabolic pathway to produce tryptophan.
• A co-repressor is required to activate a repressor in the repressible systems, or else they would always be bound.
• Enzymes E, D, C, B, and A are used to produce tryptophan.
• When we have too much tryptophan, we need to block more transcription.
• When the co-repressor (anabolic product) activates the repressor, the active repressor binds to the operator, blocking transcription so RNA polymerase cannot bind.