Molecular Biology: Transcription and Translation

Lecture 1: Structure and Function of Nucleic Acids

• To serve as genetic material, molecules must contain complex information, be capable of replication, encode the phenotype, and have the capacity to vary among species.

Types of Genetic Information

• DNA includes chromosomal DNA, mitochondrial DNA, chloroplast DNA, bacterial DNA, and viral DNA.
• RNA includes messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), ribozymes, antisense RNAs, small nuclear RNAs, and RNA Viruses.
• RNA Viruses are virulent and can reverse transcribe into DNA to be incorporated into an organism.

The Transformation Principle (Fred Griffith 1928)

• Fred Griffith studied Streptococcus pneumoniae, which causes pneumonia, in 1928.
• Griffith was able to isolate virulent and non-virulent forms of the bacteria; virulent strains cause disease, while non-virulent strains do not.
• Virulent strains evade the immune system with a slimy outer capsule.
• Non-virulent strains lack the capsule, which makes them unable to cause disease, because the immune system can recognize the antigens and release antibodies to destroy them through macrophages.
• One bacterium can transmit its characteristics to another, discovered the transformation principle.

Avery, MacLeod, and McCarty (1941)

• Avery, MacLeod, and McCarty picked up Griffith's research and purified the substance, finding it closer to DNA using enzymes.
• To prove it was DNA, they performed three enzymatic experiments on the virulent form: protease had no effect, RNase had no effect, and DNase prevented transformation.
• Published results in 1944 showed DNA's is the transformation, precipitating at the same rate also absorbing unltravoilet light as DNA.

Hershey-Chase Experiment

• Provided further support for DNA as genetic material, used T2 phage and radioactive labeling.
• T2 Phage reproduces, attaching to the cell wall to create more phages.
• Sulfur traced proteins, while phosphorus traced DNA.
• DNA, not protein, is transferred to the bacterial cell.

The Concept of the Double Helix (Chargaff's Rule)

• Chargaff's Rule shows that the percentage of adenine equals thymine [A(30.9%) = T (29.4%)] and guanine equals cytosine [G (19.9%) = C (19.8%)].
• A & G are purines, and T & C are pyrimidines.

Rosalind Franklin & Wilkins

• Already established that DNA consists of nucleotides.
• They took the first X-ray diffraction image of DNA.
• The discovered DNA is helical, contains 10 nucleotides per turn, and its helix diameter is 20 angstroms, or 2nm.

Watson & Crick (1953)

• Constructed a double-helix model based on x-ray diffraction and other known information.

Discovered:

• DNA is double-stranded and runs antiparallel.
• Complementary base pairing holds DNA and ensures a mechanism for replication.

Primary Structure of DNA:

• Sequence of nucleotides.
• Nucleotides are the monomers of nucleic acids.
• Consists of a phosphate group, nitrogenous base, and RNA or DNA sugar.
• Nitrogenous bases located on the inside of the double helix.
• Sugar-phosphate backbone held together by phosphodiester bonds.
• Nucleoside is a base and a sugar; nucleoside + phosphate group = nucleotide.

Base Pairing:

• Pyrimidine has a single ring structure and includes Cytosine, Thymine, or Uracil in RNA.
• Purine has a double-ring structure and includes Adenine and Guanine.
• DNA always pairs: G-C, A-T.
• RNA pairs: G-C, A-U.

DNA Strand & Directionality:

DNA/RNA has a 5' and a 3' end.

• The 5' carbon is linked to the phosphate group, which is linked to the 3' carbon of the next nucleotide.
• Bases are attached to the sugars and project from the side.
• There are 2 H bonds in an A-T pair and 3 H bonds in a C-G pair.

Double Helix Features:

• The nucleotides are joined by phosphodiester linkages which hold nucleotides together.
• Bases are stacked inside, and the sugar-phosphate backbone is on the outside, held together by phosphodiester bonds.
• Hydrogen bonding stabilizes base pairs.
• Major and minor grooves are sites for protein binding; the major groove is where proteins bond due to the exposed surface of nucleotides.

Configurations of DNA

• B Form: Typical configuration in vivo as proposed by Watson & Crick, forms a right-handed helix.
• A Form: A compact helix under high salt concentrations, still forms a right-handed helix.
• Z Form: A left-handed helix seen in short DNA pieces, with alternating purine/pyrimidine sequences.

RNA Structure

• Single-stranded but can fold into stem-and-loop structures, right-handed double helices, bulges, and internal loops.
• Transfer RNA (tRNA) contains anticodons and carriers for modified nucleotides.
• Characterized by an anticodon, which binds the mRNA at the ribosome, and an amino acid-carrying portion.
• Ribosomal RNA (rRNA) is essential for ribosome function.

Applications of Nucleoside Analogs

• Combat cancer and HIV by mimicking nucleotides using compounds like AZT, DDI to inhibit DNA replication of cancer cells.
• Includes compounds 3'-azidodeoxythymidine (AZT), 2', and 3'-dideoxyinosine (DDI), and Zidovudine (Retrovir, ZDV).

Mitochondrial and Chloroplast DNA

• Both are circular and encode for rRNAs, tRNAs, and essential proteins.
• Over evolution, genes appear to have transferred from the mitochondrion to the nucleus and from chloroplasts to mitochondria.

DNA vs RNA:

• All organisms use DNA as the repository for genetic information because DNA has a double-stranded structure and is resistent to enzymes.

Lecture 2: DNA Replication

• DNA Replication: Fast and Accurate
• Escherichia coli replicates 60,000 nucleotides per minute, the genome has 4.6 million bp, and replication and division occurs in 20 minutes.
• Human replicate 3,000 nucleotides per minute; the entire 3.3 billion bp genome is replicated in a few hours.
• There is 1 error per 10 million nucleotides.
• Proofreading by DNA polymerases reduces the error rate to 1 per billion nucleotides.
• There are 30 -35 new (de novo) mutations per child

Proposed Models of DNA Replication

• Conservative: Original double strand serves as a template for a new molecule.
• Dispersive: DNA breaks into fragments, serving as templates for new fragments.
• Semi-conservative: Original strand unwinds and serves as a template for a new strand.

Meselson and Stahl's Experiment

• Used nitrogen isotopes (N15 and N¹4) to distinguish old and new strands.
• Bacteria grown in N¹5 for generations make DNA heavy, then they switched to N¹4 and took samples over cell cycles.
• Collected DNA is analyzed to distinguish between strands.
• Results demonstrated semi-conservative replication as after dispersive replication you expect all DNA to be in the intermediate range with every strand having a mixture of DNA.
• Semi-conservative replication was suggested because after two rounds it would be of intemediate weight.

Modes of DNA Replication

• Theta Replication; happens in circular DNA:

  • Steps:
    • Double strand is unwinded creating a bubble during replication.
    • Replication fork, which synthises DNA in 5'3', is where the bubble grows.

• Rolling-Circle Replication; takes place in viruses and F-factor of E.coli:

  • Steps:
    • Single-strand creates a '3 and 5' group;
    • With the stand, nucleotides are added to 3'OH;
    • As the strand is elongated, the one formed is put in a plasmid.
    • The fragment that rolls can be used as a template.

• Linear Chromosomes:

  • Eukaryotes have a single replication origin and linear chromosomes.

Stages of DNA Replication

• Occurs in Prokaryotes

  • Initiation: DnaA protein binds oriC, opening the site.
  • Unwinding: Enzymes involved breaks hydrogen bonds along with other factors.
    • DNA Helicase: Breaks hydrogen bonds between bases
    • Single-Strand Binding Protein: Binds to single strands to stabilize the structure
    • DNA Gyrase (Topoisomerase): Prevents supercoiling and upstream torsional strain.
    • Topoisomerase I: Creates single-stranded breaks in DNA.
    • Topoisomerase II: Creates double-strand breaks (aka DNA gyrase) in DNA.

• Occurs in Elongation:

  • Strands are used as templates. -Primers must bind to the free 3'-OH so polymerase can bind as well. -primase is used for the bypass process -DNA polymerase replicates by using the 3'OH.

• Termination:

  • forks meet, or termination proteins (Tus) bind sequences that blocks replication.

Differences:

• Prokaryotic DNA has a single origin while eukaryotic DNA has multiple origins.
• Yeast cells replication happens at the begining with sequences (ARSs), usually rich in A-T and promotes them.

Differences in Cells Cycle:

• Prokaryotes: Continuous replication and Eukaryotes replication is controlled.

Differences Licensing:

• EUKARYOTES replication occurs at the exact origin multiple times.
• ORC authorizes to binds to genes and MCM2-7 (helicase) starts process. -The factor licensing removes can only be initiate once. -Gemin binds Cdt1 therefore degrading G!. -MCM has activity/unwids the small DNA

Differences in DNA Polymerases:

 -Alpha; synthesis initiated by primers.
 -Lagging: synthesis of strand in the correct order with the primers.
 -Esplilon: synthesis on the strand like Delta function.
 -Trans-lesion: has small pocket to dettach lesion of incorrected base.

• Nucleosome: -Complexed DNA is packaged to stabilize with histones. Replications occurs after removal of the histones. 1. Removed original form of nucleosome 2. Strands are placed and the new strand are made Telomere: -repeated at the sequences to the chromosome -acts as buffer to prevent gene loss and as DNA get shorter these area get affected first. -t' is exposed the binds. -only in somatic cells

Aging with diseases:

-shortening leads to apoptosis, when activated by cancer can leads to death.

Steps in DNA replication:

• Enzymes unravel strands and serves complementary.
• Singled strands proteins bind and stabilizes
• a '3 enzymes attach so the polymerase can synthesize
• polymerase links to '3 and can only synthesize from 5' to 3', enzymes and DNA can added. The primer is primase
• the fragments are known as okazaki when the strand starts the process and joins due to ligase.
• topisomerase unwinds helix, protein attaches, segments of the enzyme and RNA binds with the aid of polymerase.

Lecture 3: (1)Transcription and RNA Translation:

• Occurs with Gene(DNA) which makes pre-mRNA -Pre-mRNA processes splices out.

  • 5' side is is then capped and the 3' gets poly tail -Translation is used for RNA synthesis -folding localzation occurs along with other side groups -enzymatic joining help make formers.

• Transcription produce RNA template, generate proteins from cells and specific groups Both DNA strands are copied versus one strand

• Coding: template strands 3-5' sense

• Non-coding: antisense 3- 5'

• Unit; includes promoter/determinator

In bacterias

   -Machine assembles promoter
    - Consensus, recognize sequence for machines.

Eukaryotic:

-DNA RNA produced with polymerase (makes Pre-mRNA simultenously)

Termination

-separates from strain

RNA polymerase; unwids from enzyme depending where transcription ends and stop -on terminal DNA, Rho bonds where DNA is called

RNA vs terminatons

dependent; inverted repeats

• DNA are located where transcription can't bind with pronote, and other gene specifics

Eukarcityes Polymerases

• 1 Large is where rRNA is transcribed
• 2 RNA are transcribed
• 3 small and other are translated
• --RNA assembles polypeptites and bubbles during process
• Poly A tail influence the generation of transcription translation sequences

Eukaryotes

   -genes are complex, exons/introns with gene expression
   -spliced introns of RNA

Prokaryotes:

   -a sequence is on the ribosome helps stabilize what gets translated and time it. Extensively happen nucleus towards cytoplasm.

Processed extensively in the Prokayotes 5' of mRA stabilizes, there is the DNA and the bond linkage from the triphosphate that takes process a tail attaches. alternative to allow chains of DNA to be flexible from degermation, is a process from a form of mRNA in order to enable it. editing: what bind enable edits to to modify/switch from Dna

Lecuture 4: (2) Translation and Post Translaitonal Processing

• Proteins: are amino held in peptide bonds in groups such as carboxyl.
• Determines the nucleotides for amino acids*
• 3 nucleotides encoding a single acid. 4 locations, coding for 20 differnt aicd.
• Sence/stop condoms for UAA, UAG, UGA. Some can can be indicated by more! Amino Translation.
• RNA function as acids translations, the mRNa can be the tRNA and vice versa! that is very important!

Aminoacyl

20 can be specific for proteins through tRNA. With the 3rd base and hydrogen bond is the codon, requires lest energy to get together, by according to wattston.

• -A frame and what the start condom allows for 1 frame to be allowed. Ribosome: in bacterias when subunits of a small bonds occurs. start tNA attachees DNA helps the subnit in 5 to recognize as it atatches when it's made.

Eukaryotics:

5' cap and when the sequences indentifies at the site for codons to be used from Marylyn.

Polypepties:

B. the ribosome stops the codon Factor: help detach in its entirety, hydrolysis will ccur when mRAN is gone. When is over mutated and the protein isn't made there is the decay the aids stop to be complete if not there the STOP isn't finished Mechanism: if what ever not encountered it signals to degrand.

Protein:

can form for 3-20 sugars and for hemoglobin to increase their surface. subunits can be monomers for idenitical proteins

Post translatnioal

Lecutre 5:1 Gene Regulations in Prokaryotes:

• regulation for genes. A system for when its turn off/one when needed, multiple level regulation

• Regulatory elements DNA, regulates not transcribed.

• Positive controls/ increase expression

• negative decrease Binding of Element when binding it has a effect on sugar it can't reverse. OPerans isn't typicial with what get control. it binds an operate where location is the the polymerase, types is a repressor can inhibit and stimulate

controls are operated with sequence promoter. Trans; activate/repress when on the chmromosome. polymerase.

Lec Operational Components

RNA polymeran/ expressors is where one gene has copies to copy! Trans factors molecules for regulatin. simplified what does components: where allactose lactose structural when genes bond in all factor. repessor will prevent operon! is there but the repressors will start over! active protein is there from constituve to allway productors and Monads using mutation to do that. is also reguylator gene, it can happen like with what copy it does is sufficent! 1 will sufficent! mutation for transcription which mean non futioning and polymerase won'g be abkle to biold!

"Super can't be activated/repressed/ operon" is where this goes

Lecture 6: (2) Gene Regulation in Prokaryotes

• A worm up for lactose. when is glucose present and there other proteins to process then it gets repressed and it's a hunger. bind where the RNA is afiity but need to bind CAMP first! or else it will be less function: repressor

• the more glucoe the higher is the functions. e coll synthesizes

• Tryptoman is expresed or high

• operon: tryptophan the gene to bind with operator but gets transcribed and also contains 2 when its low, allows enablings. turns but controllings what going. what bonds with RNA to regualte.

RIBOSOMES that affect mRNA stucture:

-if the Glcm is absent then it transcribes more for GICM btu if it is more present then it can not transcribe. mRNA and it gets deagraded!

LECTURE 7: (1): GENE REGUALTION IN EUKARYOTES:

• how many do not work or relate with complexity and how they re used.
• House always atice where the gly colysys what actviates based on if the expression happen at the cell. it all depds! biochemical express level it varies depending on the activity for people and and the genetic traits!

REgulatotry genes interaction:

A. DNA structure, promoter. regulators bind and alter DNA, stable form 2. DNA packigin how they bind and with whay 4 stabilty

• factors bind and and allow polymarase with minimal activity, transduction allow transcription
• proteins wrap in histones. where gene represses and can't have activate depending on sites which where the chargde occur

Q: Dna: the methyltion. methyl proteins occur there, the coordinate where regulation is allowed or repress them and insulator is where it acts.

LECTURE 8:"2"

• Regualiton how RNA are degraded the gene the exons link to create and make vary proteins. what is can get spliced the more and it regulates. the genes produce different levels! Ribsnuceleases destroy and degrade! the removal of the Cap poly is what destablizes the cell. and interferes also joins in the pressor. mRA are regualte with express.

SI are introduced in where mirna are transdcribed a how rna interference: inhibits the translation. Q; what deactivates how it's affected

WHAT ARE ALL IN DNA? A; COMMON REGIONS HELP prevent where methyltaied DNA

• Honeybee caste difference, royalty is silenced. XisT RNA helps coat one
• transuma , increased lick to help offpsring

Epi how it is related to differentation and the 3 the process work to the way methylation can regulate that!

Explore the core concepts of transcription, translation, and gene regulation, including transcription phases, tRNA function, and mRNA processing. Learn about operons, gene expression, and post-translational modifications.