Central Dogma of Life & DNA Replication PDF

#**The Central Dogma of Life** - The Central Dogma holds that genetic information is expressed in a specific order, from DNA to protein synthesis. ## **DNA Replication** - DNA replication is the process of making copies of DNA. - A single strand of DNA serves as a template for a new strand. - The...

#**The Central Dogma of Life** - The Central Dogma holds that genetic information is expressed in a specific order, from DNA to protein synthesis. ## **DNA Replication** - DNA replication is the process of making copies of DNA. - A single strand of DNA serves as a template for a new strand. - The rules of base pairing direct replication. - DNA is replicated during the S (synthesis) stage of the cell cycle. - Each body cell gets a complete set of identical DNA. ## **Models of DNA Replication** - There are three (3) Models of Replication: - **Semiconservative**: each daughter has 1 parental and 1 new strand. - **Conservative**: 2 parental strands stay together. - **Dispersive**: DNA is fragmented, both new and old DNA coexist in the same strand. ## **Semiconservative Replication** - The process of DNA replication is called 'semiconservative' replication. This means that in each new double helix of DNA, one strand was from the parent. ## **Meselson and Stahl Experiments** - **Predictions**: - **Conservative**: The original strands stay together. - **Semiconservative**: Each new strand is half from the parent, half new. - **Dispersive**: New strands are a mixture of old and new. - **Results**: - First replication: One band in the middle. - Second replication: Two bands, one in the middle, one near the bottom. - **Conclusion**: Replicated DNA formed one band in the middle after one replication, and two bands (one denser than the other) after two replications. ##**Assignment II** - **A.** Describe the Meselson and Stahl experiments in detail. - **B.** What would be the outcome if the experiment continued for: - i. three generations - ii. four generations - iii. five generations ## **DNA Replication Enzymes and Their Functions** - **Helicase**: Unwinds the parental double helix at replication forks. - **Single-strand binding protein**: Binds to and stabilizes single-stranded DNA until it is used as a template. - **Topoisomerase/DNA Gyrase**: Relieves overwinding strain ahead of replication forks by breaking, swiveling, and rejoining DNA strands. - **Primase**: Synthesizes an RNA primer at 5' end of the leading strand and at 5' end of each Okazaki fragments of lagging strand. - **DNA pol III**: Using parental DNA as a template, synthesizes new DNA strand by adding nucleotides to an RNA primer or a pre-existing DNA strand. - **DNA pol 1**: Removes RNA nucleotides of primer from 5' end and replaces them with DNA nucleotides added to 3' end of the adjacent fragment. - **DNA ligase**: Joins Okazaki fragments of lagging strand, on leading strand, joins 3' end of DNA that replaces primer to rest of leading strand DNA. ## **Replication Fork** - As the double helix unwinds, the two complementary strands of DNA separate from each other and form a Y-shaped structure known as the replication fork. ## **DNA Replication: leading strand** - **RNA primase enzymes** begin the replication process by building a small complementary RNA segment called **RNA primers (10-60 ribonucleotides long)**. - **DNA polymerase III** begins to add DNA nucleotides to the primer. - Since **DNA polymerase III** only builds in the 5'→3' direction, the two new strands begin to be assembled in opposite directions. - **DNA polymerase III** is able to continue continuously. - No need for the **RNA primase** to add additional primers. This is called the **leading strand.** ## **DNA Replication: lagging strand** - On the opposite strand, **DNA polymerase III** is moving away from the replication fork (**lagging strand**). - **RNA primase** attaches another primer allowing **DNA polymerase III** to begin from a new point. - The pattern created on the second strand is a series of RNA primers and short DNA fragments, called **Okazaki fragments**. - **DNA polymerase I** removes the RNA nucleotides and replaces them with DNA nucleotides. - **DNA ligase** catalyzes the formation of phosphodiester bonds to seal the strand. ## **A General Model for DNA Replication** 1. The DNA molecule is unwound and prepared for synthesis by the action of **DNA gyrase**, **DNA helicase** and the single-stranded DNA binding proteins. 2. A free 3' OH group is required for replication but when the two chains separate, no group of that nature exists. **RNA primers** are synthesized, and the free 3' OH of the primer is used to begin replication. 3. The replication fork moves in one direction, but DNA replication only goes in the 5' to 3' direction. This paradox is resolved by the use of **Okazaki fragments**. These are short, discontinuous replication products that are produced off the lagging strand. This is in comparison to the continuous strand that is made off the leading strand. 4. The final product does not have RNA stretches in it. These are removed by the 5' to 3' **exonuclease** activity of Polymerase I. 5. The final product does not have any gaps in the DNA that result from the removal of the RNA primer. These are filled in by the 5' to 3' polymerase action of **DNA Polymerase I**. 6. **DNA polymerase** does not have the ability to form the final bond. This is done by the enzyme **DNA ligase**. ## **Checking for Errors** - **DNA polymerases** that carry out replication also play another important role. - As they assemble new DNA strands, they **proof-read** and **correct** errors (base-pair mismatches). - **Proof-reading**: While creating the complementary strand, if a mismatch occurs, **DNA polymerase III** may back up, repair, and continue. - **Repairing**: **DNA repair mechanisms** of **DNA Polymerase I and II** may locate distortions in the strands between replication events and remove a piece of the strand, **DNA polymerase III** will fill the gap, and **DNA ligase** will seal the strand. ## **Proof-reading** - **DNA polymerase III** continues adding nucleotides in the forward direction. - If the enzyme adds a mismatched nucleotide, the enzyme acts as an **exonuclease (cleave nucleotides)** to remove the mismatched nucleotide. - The enzyme resumes activity as **DNA polymerase**. ## **Repairing** - **DNA polymerase II** repairs damage to DNA that occurs between replication events. - **Repair complexes** remove several to many bases, leaving a gap in the DNA. - Gap is filled in by a **DNA polymerase**, using the template as a guide. - Nick is sealed by **DNA ligase** to complete repair. ## **Assignment III** - Compare and contrast DNA replication on the leading and lagging strands. ## **Transcription: DNA to mRNA** - **Transcription** is the process by which an RNA sequence is produced from a DNA template. - **RNA polymerase II** begins RNA synthesis at the transcription start point, which has the sequence **TATAAA (TATA box)**. - Only one of the two **DNA strands** is copied into an **mRNA strand** during transcription. - The strand that gets transcribed is the **template** or **antisense strand**. - The **RNA strand** is made in the 5'→3' direction using the 3'→5' **DNA strand** as a template. - **Nucleotides** are added into a complementary strand of **mRNA** based on the **DNA code**. ## **Types of RNA** 1. **Messenger RNA (mRNA)** - carries DNA's message from the nucleus to the ribosome. 2. **Transfer RNA (tRNA)** - carries the correct amino acids to the ribosome so they can be added to the growing protein chain. 3. **Ribosomal RNA (rRNA)** - makes up part of the ribosome. Helps read mRNAs message and assemble proteins. ##**Transcription Process** - In the initiation process, the DNA is unzipped by **RNA polymerase**. - The newly formed **mRNA** moves out of the nucleus to **ribosomes** in the **cytoplasm** (for translation) and the DNA re-winds. ## **Translation: mRNA to Proteins** - **Translation** is the process of protein synthesis in which the genetic information encoded in mRNA is translated into polypeptide chains of amino acids. - Once the DNA has been transcribed to mRNA, the codons must be translated to the amino acid sequence of the protein. - The first step in translation is **activation** of the **tRNA**. - Each tRNA has a triplet called an **anticodon** that complements a codon on mRNA. ## **Translation Process** - **Initiation** of protein synthesis occurs when an mRNA attaches to a ribosome. - On the mRNA, the **start codon (AUG)** binds to a tRNA with methionine. - The second codon attaches to a tRNA with the next amino acid. - A **peptide bond** forms between the adjacent amino acids at the first and second codons. - The first tRNA detaches from the ribosome and the ribosome shifts to the adjacent codon on the mRNA (this process is called **translocation**). - A third codon can now attach where the second one was before translocation. - **Amino acids** are carried by **transfer RNA (tRNA)**. - The **anticodons** on tRNA are complementary to the codons on mRNA. ## **Translation Process** - After a polypeptide with all the amino acids for a protein is synthesized, the ribosome reaches the "**stop**" codon - UGA, UAA, or UAG. - There is no tRNA with an anticodon for the "**stop**" codons. - Therefore, protein synthesis ends (**termination**). - Finally, the protein is shipped to the **Golgi body** where it is altered and shipped to where its destination. - At its final destination, the protein will perform its specific function. ## **Genetic Code** - The genetic code is the set of rules by which information encoded in mRNA sequences is converted into proteins (amino acid sequences) by living cells. - It consists of sets of three nucleotides (triplets) in mRNA called **codons** that specify the amino acids and their sequence in the protein. - **Codons** are a triplet of bases which encodes a particular amino acid. - The codons can translate for **20 amino acids**. - Codons of three bases on mRNA correspond to one amino acid in a polypeptide. - As there are four bases, there are 64 (43) different **codon combinations**. Of these, 61 code for the 20 amino acids, 3 code for **stop codons**. ## **Genetic Code** - Different codons can translate for the same amino acid (e.g. GAU and GAC both translate for Aspartate) therefore the genetic code is said to be **degenerate**. - The order of the codons determines the amino acid sequence for a protein. - The coding region always starts with a **START codon (AUG)** therefore the first amino acid in all polypeptides is Methionine. - The coding region of mRNA terminates with a **STOP codon (UGA, UAA, or UAG)**. The **STOP codons** do not add an amino acid. Instead, it causes the release of the polypeptide. ## **The Genetic Code** This table shows the genetic code, which specifies which amino acid each codon encodes. | First base of codon | Second base of codon | | | | |---|---|---|---|---| | U | C | A | G | | | U | | | | U | | U | | | | C | | U | | | | A | | U | | | | G | | C | | | | U | | C | | | | C | | C | | | | A | | C | | | | G | | A | | | | U | | A | | | | C | | A | | | | A | | A | | | | G | | G | | | | U | | G | | | | C | | G | | | | A | | G | | | | G | ## **Features of the Genetic Code** - The genetic code - is a triplet code - is universal (exceptions exist) - is commaless - is degenerate/redundant - has start and stop signals - nonoverlapping ## **Protein Synthesis** 1. First transcribe the DNA code into its **mRNA**. Do this by complimentary base-pairing (A-U, G-C). 2. **Next**, break the **mRNA** into codon or three letters. 3. Plug the codons into the chart and find the amino acids. ## **Protein Synthesis** | DNA Triplet | mRNA Codon | tRNA Anticodon | Amino Acid (and/or instruction) | |---|---|---|---| | AAA | UUU | AAA | Phenylalanine | | AAT | UUA | AAU | Leucine | | ACA | UGU | ACA | Cysteine | | CAA | GUU | CAA | Valine | | GGG | CCC | GGG | Proline | | CGA | GCU | CGA | Alanine | | TAC | AUG | UAC | Methionine; start codon | | ATT | UAA | [none] | Stop codon | ## **Haemoglobin Mutations** | | DNA | mRNA | Amino Acid | Properties of AA | Effect on protein | Disease | |---|---|---|---|---|---|---| | Original codon 6 | CTC | GAG | Glutamic Acid | Hydrophilic | Normal | None | | Mutation 1 | CTT | GAA | Glutamic Acid | Hydrophilic | Neutral | None | | Mutation 2 | GTC | CAG | Glutamine | Hydrophilic | Neutral | None | | Mutation 3 | CAC | GUG | Valine | Hydrophobic | Loses water solubility | Sickle Cell Anaemia |