DNA Replication and Repair

DNA as Hereditary Material

• DNA structure has a double helical confirmation
• Key concepts include genome, gene, karyotype, and complexity
• Higher level DNA packaging involves chromatin, nucleosomes/histone proteins, chromosomes, and heterochromatin-euchromatin

DNA Replication

• DNA replication is semiconservative
• Involves replication forks, Okazaki fragments, and primers
• DNA has proofreading capabilities

DNA Repair

• Repair is completed using Nucleotide Excision Repair, Mismatch Repair, and Nonhomologous End Joining

Decoding Genetic Instructions in DNA

• In the 1950s, scientists determined that hereditary information is encoded in the linear sequence of nucleotide subunits in DNA
• Genetic instructions use a four-letter alphabet to direct forming the building blocks of organisms
• Cells use the information genes contain to direct the synthesis of protein
• Proteins are the primary building blocks of cells that determine cell structure and function
• The sequence of the 20 different amino acid subunits in its polypeptide chain determine the properties and function of a protein molecule
• Genetic instructions specify the amino acid sequences of proteins

Central Dogma

• DNA does not synthesis proteins but delegates tasks to a team
• Ribonucleic acid (RNA) copies the nucleotide sequence of a DNA segment when the cell needs protein
• A gene is a segment of DNA, and the cell uses resulting RNA copies to direct the synthesis of protein
• Genetic information flows from DNA to RNA to protein
• Transcription is copying DNA into RNA
• Translation is using RNA information to make protein

Transcription and Translation

• Cells read out or express genes using transcription and translation
• Identical RNA copies can originate from the same gene, and each can direct identical protein molecules for synthesis
• Successive amplification enables cells to rapidly synthesize large amounts of proteins when needed
• Gene expression is dependent on how fast the first mRNA copy has been made
• Gene expression is the regulation for each gene based on its demand

RNA differences compared to DNA

• The first step is to copy the nucleotide sequence of a gene into RNA
• Transcription is the process of copying information into another form while keeping the same language of nucleotides
• RNA, like DNA, is a polymer made of four different nucleotide subunits, linked by phosphodiester bonds
• RNA differs chemically as the nucleotides in RNA are ribonucleotides, containing the sugar ribose
• RNA has the bases adenine (A), guanine (G), and cytosine (C), like DNA, but includes uracil (U) instead of thymine (T)
• RNA contains uracil (U) that can base-pair by hydrogen-bonding with A
• RNA is single-stranded with the ability to fold into various shapes (similar to a polypeptide chain), unlike double-stranded DNA
• Folding into a complex three-dimensional shape enables RNA to provide more information to cells than solely conveying information between DNA and protein
• RNA can have structural, regulatory, or catalytic roles, whereas DNA functions solely as an information store

Transcription process

• Transcription synthesizes RNA with certain similarities to DNA replication
• The opening and unwinding of a small DNA double helix exposes the bases on each DNA
• One DNA strand acts as a template for RNA synthesis
• RNA nucleotides are added one by one to the growing RNA chain
• The nucleotide sequence of the RNA chain is determined through complementary base-pairing with the DNA template
• The enzyme RNA polymerase covalently links the incoming ribonucleotide to the growing RNA chain when a good match is made
• The RNA transcript has a nucleotide sequence exactly complementary to the DNA strand used as the template and elongates to 3' end
• Unlike newly formed DNA strands, the RNA strand does not remain hydrogen-bonded to the DNA template; instead, the RNA chain is displaced behind the region where ribonucleotides are added, and the DNA helix re-forms
• Transcribed RNA molecules are single-stranded
• RNA molecules are shorter than DNA molecules because RNAs are copied from only a limited region of DNA

RNA Polymerase

• RNA Polymerases catalyze formation of phosphodiester bonds (like DNA polymerase) to link nucleotides and make the sugar-phosphate backbone
• RNA polymerase moves stepwise along DNA, unwinding the DNA helix for complementary base-pairing
• It extends the growing RNA chain one nucleotide at a time in the 5'-to-3' direction
• Incoming ribonucleoside triphosphates (ATP, CTP, UTP, and GTP) provide the energy for reaction

Differences Between Enzymes

• RNA polymerase uses ribonucleoside phosphates as substrates for catalyzing linkage of ribonucleotides and not deoxyribonucleotides
• RNA polymerase can initiate an RNA chain without a primer, unlike DNA polymerase involved in DNA replication
• Transcription doesn't require high accuracy
• RNA polymerase has about one mistake per 104 nucleotides in copying RNA
• DNA polymerase has about one mistake per 107 nucleotides copied

RNA Types

• Most genes in a cell's DNA specify the amino acid sequences of proteins
• RNAs that encode proteins are called messenger RNAs (mRNAs)
• The final product of other genes is RNA that serve in regulatory, structural, and catalytic components of cells

Nonmessenger RNAs

• Ribosomal RNAs (rRNAs) form ribosomes, which translate mRNAs into protein
• Transfer RNAs (tRNAs) act as adaptors, selecting specific amino acids for ribosome use
• MicroRNAs (miRNAs) regulate eukaryotic gene expression

Promoters in Prokaryotes

• Transcription initiation is main point where cell selects proteins or RNAs to be produced.
• RNA polymerase needs to recognize gene start and bind to DNA
• Sequence of nucleotides called promoter is needed for bacteria and eukaryotes upstream from start point
• Bacterial trancription is simpler than eukaryotic transcription

Bacterial RNA Polymerase

• When a bacterial polymerase collides with a molecule of DNA it weakly binds, then slides along its length until it encounters the promoter where it binds tightly.
• After binding, the bacterial RNA polymerase will open the DNA double helix in front of the promoter to expose nucleotides.
• As incoming ribonucleoside triphosphates base pair with complementary nucleotides the polymerase joins two of them to make its RNA chain.
• Chain elongation continues until the enzyme reaches a second signal in the DNA called the terminator where the polymerase releases the DNA template and RNA transcript.
• The terminator sequence is located inside the gene and becomes part of the 3' RNA end
• Bacteria use the sigma (σ) factor, a subunit of RNA polymerase, to recognize the promoter sequence on the DNA
• RNA needs to determine which of the two DNA strands to use as a template for transcription, thus every promoter contains a certain polarity
• Once the enzyme has positioned itself to synthesize RNA in the 5' to 3' direction the polymerase must use the DNA strand oriented in the 3' to 5' direction as its template
• With respect to the chromosome as a whole, the direction of transcription varies from gene to gene

Bacterial Promoters and Terminators

• The green region specifies a promoter, with +1 designating the first nucleotide transcribed
• The promoter's polarity determines polymerase orientation, coding -10 and -35 contain DNA sequences like those shown above
• Red regions signal an RNA polymerase to terminate transcription
• Promoter nucleotide sequences are not transcribed while terminator nucleotide sequences are
• Conventionally the gene sequence is the non-template strand because it has the same sequence of the transcribed RNA (with T substituting for U)

Eukaryotic Transcription

• Transcription initiation differs from Eukaryotes
• RNA Polymerase (I,II,III) transcribe different genes
• RNA Polymerase I+III transcribe transfer RNA
• RNA Polymerase II transcribes majority if eukaryotic genes
• Eukaryotic RNA polymerase requires accessory proteins called transcription factors
• Control of initation are more elaborate than in prokaryotes
• Individual genes are spread out along the DNA
• Regulatory DNA enable Eukaryotes to have higher transcriptional regulation form
• Eukaryotic transcription initation takes into account for the packing of DNA into nucleosomes

Eukaryotes vs Bacteria

• Eukaryotic transcription requires transcription factors, whereas bacterial RNA can initiate it on its own
• Eukaryotic transcription is more elaborate than prokaryotes
• Spreading out along DNA creates regulatory controls that enable higher transcriptional forms
• In bacteria promoters allow positioning
• Eukaryotes must initiate transcription

TATA Box

• The complex of general trancription factors starts with TFIID binding a short DNA double helix known as the TATA box
• TFIID causes a DNA distortion that serves as landmark for other protein assembly at the promoter
• TATA box promoters used by RNA polymerase II are located 25 nucleotides upstream from the transcription site
• Following TFIID binding, other factors assemble for transcription
• After RNA polymerase II has been positioned, it needs to be released from the general transcription complex to make RNA
• The general transcription factor TFIIH (contains a protein kinase subunit) aids this release through addition of phosphate groups to its "tail”.
• Transcription factors dissociate and become available to initiate a round of transcription with a new RNA polymerase molecule after it’s begun
• When RNA polymerase II finishes it is released.
• Only dephosphorylated RNA Polymerase II is able to initiate RNA synthesis

Eukaryotic Transcription

• Transcription into RNA templating is the same in all organisms
• RNA transcript handling before the cell to make protein differs due to Bacteria having exposed Cytoplasm while Eukaryotic are enclosed in a Nucleus
• In bacteria, ribosomes can immediately attach to RNA
• In Eukaryotic cells, DNA is enclosed within a nucleus, Transcription takes place in nucleus and synthesis takes place on ribosomoes in cytoplasm
• Transcripted Protein must move out of nucleus and into nuclear envelope

mRNA Processing

• Eukaryotic RNA must move through RNA processing
• The process consist of "capping", "splicing", and "polyadenylation"
• These take place as the RNA transcribes
• Enzymes involved in RNA "ride on" the tail and process the transcript

Two Processing Steps

• Capping and Polyadenylation happen only on mRNA
• RNA Capping changes to the 5' end when the guanine (G) nucleotide bearing a methyl group is added in a different way after RNA Polymerase 2 has produced 24 nucleotides
• Polyadenylation provides a new transcription mRNA with a new 3' end structure and the enzyme will add a series of adenine (A) nucleotides that is a poly-A tail that can be hundreds of nucleotides long.
• Capping and Polyadenylation increase stability, facilitate export, and marks the molecule
• It also makes sure that both ends are present and that the message is completed before protein synthesis

Introns

• Addition processing has to happen for mRNA to function
• It's caused by a surprising feature of most Eukaryotic genes
• Most proteins are encoded by a DNA stretch that becomes an uninterrupted mRNA sequence
• Eukaryotic genes coding sequence have long sequences called introns that interrupt coding
• Also has pieces of sequence called scattered sequences that represent only a fraction of total length

mRNA process

• During mRNA creation, the entire gene is transcribed into RNA
• Process of RNA splicing begins to remove Introns as RNA polymerase II
• An mRNA is produced when a poly A tail is received by the transcript after it's sliced and modified mRNA is able to leave the nucleus and be transcripted to protein

Alternative Splicing

• Alternative Splicing has benefits
• Transcripts can use splicing in different ways, with 95% of human genes thought for undergo this
• It can allows differing proteins to be produced depending on gene and allow to rise for novel proteins

Transport

• Eukaryotic Pre-mRNA synthesis and processing happen in the cells nucleus
• Mature mRNA is the only able to be transported from the nucleus to cytosol
• Nucleur complexes act as gates to move large molecules and connect nucleoplasm with cytosol
• To be "export ready" mRNA needs to bind in orderly way to proteins that show message is completed
• Proteins like poly-A-binding protein, cap-binding, and spliced proteins need to bind to mRNA correctly.

mRNA Waste

• "Waste" RNAS that are stuck in the nucleus can re-used blocks for transcription again mRNA life time
• mRNA can be degraded by ribonucleases (RNAses) but depending on sequence can change
• Affects the protein it produces
• Proteins are in part controlled by nucleotide in the portion of RNA
• Lifetimes can help control the amount of protein that can be synthesized

Translation

• Transcription has a mean that can transfers information easily
• DNA and transcription have similar structures
• It's transferred into more simple forms that related
• Translation has information that can use symbols
• It has "rules to the sequence" that makes a protein for a "genetic code"

mRNA Sequences

• The nucleotides in an MRNA molecule need to be read consecutively in the amount of 3
• Three consecutive nucleotides and can specify an "codon" that codes information of acid
• 4"x"4"x"4= 64
• Proteins have only 20 acids and the rest are from codons
• Organisms use present day genetic code

Reading Frame

• Nucleotides in the MRNA are reed from 5 to 3
• It can vary based on the sequence and type
• There can be a few frames when decoding starts
• It is specific to the protein and coding needs

Transfer RNA

• MRNA Molecules don't need to specify to acid
• It has a protein adaptor that bind a codons to a side of protein
• Transfer RNAs a set of molecules that equal to about 80 in length
• Fold back on the self which resemble on cloverleaf

tRNAs

• Has a function for proteins
• The anticodon is is nucleotides that bind thought base with complimentary molecule
• Single Stranded region at 3 is a where acid is attached covalently

Molecule Roles

• In order to be an adaptor each must be "charged" to the acid correctly with aminoacyl-tRNA synthases to couple with correct molecules
• There exist 20 Synthetases for acids that act as anticodon arm on the correct Molecule

Complementary Roles

• tRNA molecules recognize a codon with the same base-pairing as transcription
• Molecular machine can move molecule to quickly to mRNA
• Can capture, holds, then the acid linked to the polypeptide Chain
• Done in prokaryotes and eukaryotes
• A large complex that made into dozens small proteins that is ribosomal
• A typical eukaryotic cell contains millions on its cytoplasm Ribosomes
• Structure and function are similar
• Composed of 1 large small Subunit to create a complete ribosome
• Small matches rRNA on MRNA while large bonds

Binding

• Come to gather 5 ends with protein
• To 3 direction
• Nucleotide sequence transfers it
• Added in correct molecule chain
• Finished then separate

Ribosomes Site

• Each binding site of has molecule and 3 TRNA sides
• A PE are the molecules in AminoacyltRNA
• For adding they enter with compliments
• It acids with polypeptide chain held with P
• Re-actions happen and added acid polypeptide
• It can add to protein growing in amino its carboxyl until a molecule is encountered

Steps

• 1 has charged TRNA and aids in the ribosome
• Molecule can base-pair or acid can be paired
• Position is preserved with the synthesis
• Chain from T site is joins the side
• Translocated to units and the subunit is shift
• Chain moves side is now translated
• Empty side is now reset

Initiation

• Translation requires a start signal
• Site synthesis that beings on mRNA
• Error can result if read wrong in the start
• MRNA "can" but isn't with amino acids

Eukarytoes Translation

• Initiator Trna is charges
• Added and called is
• Not normal and and cant bind normally
• Has all and binds in absence with all
• Molecule mRNA is at 5 ends
• Goes forward and searches for AUG
• Recogniton releases all factors and bind

End of Translation

• A signal with one
• They re not recognized
• Doesn’t special but signal can only stop to ribosome
• Binding alters
• Also and water with molecules instead for acids
• Chain and cell are released
• Disassociates to molecules now to begin