2nd Exam - ABT 101

Central Dogma of Molecular Biology

• detailed residue-by-residue transfer of sequential information

Deoxyribonucleic Acid (DNA)

• semi-conservative

• bidirectional

• leading and lagging strand

• replication fork

helicase

unwound DNA by breaking down hydrogen bonds between nucleotide in base pair

single-strand binding protein (SSB)

stabilize unwound strands so they wont bind again

topoisomerase/gyrase

relieves supercoiling

DNA primase

stretch of RNA attaches to this

RNA primer

attaches to DNA primase to provide a free 3′-OH group required by DNA polymerase to start DNA replication

DNA Pol III

• synthesizing new DNA strands in 5’ to 3’ direction

• possesses 3’→5’ exonuclease (free nucleoside triphosphates) activity for proofreading

Okazaki fragments

discontinuous segmentsd

DNA ligase

glues/bonds adjacent nucleotides that are not held together by phosphodiester bonds (between fragments)

DNA Pol I

• remove RNA primers and replace with correct nucleotides (proofreading)

DNA Pol II

• backup and repair enzyme

genes

• ‘beads on a string’

• basic unit of info

gene linear arrangement

UPSTREAM

• enhancer - binds proteins to boost gene transcription

• recognition site - where protein will bind

• regulatory region - regulate the expression of gene

• repressor - blocks transcript

• promoter - RNA pol binding site that initiates transcription

DOWNSTREAM

• transcription start site (TSS) - loc where transcription begins (+1 position - ATG)

• 5’ UTR - non-coding that control how, when, and how much protein the cell makes from that mRNA

• signal peptide sequence - dictates protein transport to continue the expression

• exons - coding region for proteins and kept for the final mRNA as they contain information for making proteins

• introns - non-coding for proteins that organize and regulate those instructions but are spliced out during RNA processing (removed during splicing)

• terminator site - signal RNA pol to cease transcription

• 3’ UTR - to control gene expression by regulating mRNA stability, translation efficiency, and subcellular localization

• poly A signal - prevents early decay of mRNA (decay = lesser proteins)

introns

intervening sections that are removed but can help regulate the gene and allow different exon combinations

exons

useful sections that are exported/expressed in the mature mRNA

mRNA

codes for protein

rRNA

forms basic structure of ribosomes and catalyze protein synthesis

tRNA

central to protein synthesis as adaptors between mRNA and amino acids

promoters

PROKAYOTES

1. pribnow box (-10 element) - TATAAT

2. (-35 element) - TTGACA (eg. e. coli, lacto bacillus)

EUKARYOTES

1. TATA box (-25-30 element) - TATAAA(A) (eg. plant)

DNA binding motifs

Help proteins attach to specific parts of DNA to control gene expression

1. zinc finger - zinc ion (Zn²⁺) to stabilize its structure

2. helix-turn-helix - two α-helices connected by a turn

3. leucine zipper - Two α-helices zip together through leucine interactions, forming a dimer

DNA Supercoiling

as RNA pol proceeds to transcribe the DNA strand, it results to squeezing tension and untwisted strands

Topo Type I & II - snip to fix then glue

types of RNA polymerase

1. Polymerase I - makes rRNA

2. Polymerase II - makes mRNA

3. Polymerase III - makes tRNA

RNA Polymerase II

1. carboxy terminal domain (CTD)

   • addition of phosphate groups

     • phosphorylation causes disconnection of polymerase from transcription factors (proteins that bind to specific dna sequence) to move and build the mRNA

2. newly phosphorylated tail hold the three major mRNA processing teams

   • capping

   • splicing

   • polyadenylation

capping

• addition of 5’ cap to pre-mRNA

• protects RNA and helps with translation

• methyl group is added to the guanine nucleotide at the 5’ end

• cap binding complex (CBC) proteins - also protect the cap then later direct transcript its exit from nucleus

polyadenylation

• addition of poly(A) tail to the 3’ end

• increases mRNA stability and protects from degradation

splicing

• introns are removed while exons still intact

• produces mature mRNA

• begins with GT (5’ splice site) and ends with AG (3’ splice site)

• branch site (A) helps form the lariat loop (shape made when the intron is removed)

GT-A-AG

cutting - GT loops around A then cut at AG

spliceosome

• multi-protein and RNA machine that performs splicing

• made of five snRNAs (U1, U2, U4, U5, U6) combined with proteins to form snRNPs

U1 - binds to GU

U2 - binds to A

U4/5/6 - trimer (U5_5’ exon, U6 binds to U2)

Cell Avoids Splicing Mistakes

• introns very large, while exons are small (around 150 bases long)

  • exons are easier to spot

• exon definition to mark the pieces

RNA processing

heterogenous nuclear RNA (hnRNA) —»»» mature mRNA

Transcription terminators

• specific DNA sequence near the end of a gene that tells RNA polymerase to stop transcription

1. rho-independent termination

   • no rho protein

   • RNA → 🪮 hairpin forms → RNA polymerase pauses → RNA is released

2. rho-dependent termination

   • rho protein - moves along the newly made RNA then eventually catches up with RNA polymerase

   • causes RNA-DNA interaction to break

tRNA

• anticodon - 3’ to 5’ sequences that complements mRNA (codon)

• acceptor arm - amino acid code on 3’ end

• T and D loops - structure for interface w/ aminoacyl-tRNA synthetase

  • wobble position - 3rd base meets the 1st base of anticodon

ribosome

• cell's protein-building factories

• large subunit(LSU) and small subunit (SSU)

PROKARYOTIC RIBOSOME (70S total)

1. 50S LSU

   • 5S rRNA

   • 23S rRNA

2. 30S SSU

   • 16S rRNA

EUKARYOTIC RIBOSOME (80S total)

1. 60S LSU

   • 5S rRNA

   • 28S rRNA

   • 5.8S rRNA

2. 40SSU

   • 18S rRNA

LSU sites

1. A site (Aminoacyl) - next incoming tRNA enters

2. P site (Peptidyl) - growing protein/polypeptide chain

3. E site (Exit) - empty tRNAs exit the ribosome

Translation Elongation

EF-Tu + GTP — deliver new tRNA to the ribosome bound to a high-energy molecule (GTP)

built-in delay and hybrid holding step boost translation accuracy

wrong match — tRNA binds weakly, slips out, and dissociates before any energy is used

correct match - strong base pairing triggers EF-Tu to split GTP into GDP and phosphate (P), EF-Tu changes shape and detaches from tRNA

Translation terminators

• stop codon - UAA, UAG, UGA

• no tRNA binds to this set of codons

• while in A site, release factors will appear

  • ribosome adds water to the last peptide (carboxyl end)

Post-Translational Modification (PTM)

• chemical tag (eg. phosphate,methyl, or sugar grps) attached to a protein after it has been built by the ribosome to change its shape, location, or job

• Proteome - complete set of functional, active proteins in a cell

• Genome - Your complete set of DNA/genes (the recipe library)

1 GENE = receive many diff PTMs = Proteome larger and more complex

• protein folding as they are translated to interact with other molecules

four possible pathways of new proteins

1. Folds Automatically (No Help Needed)

2. Folds with a Helper (Molecular Chaperones)

3. Destroyed and Recycled (The Proteasome)

4. Clumping Up (Protein Aggregates) - misfolded proteins

Molecule Chaperones

• elper proteins that act like "babysitters" or "folding assistants" to make sure other proteins fold correctly instead of clumping into toxic messes

1. Hsp70 — grabbing raw, uncoiled amino acid chains as they are being made Prevents early clumping)

2. Hsp60 — like an isolated barrel or chamber for final folding checks (Quality control & cell survival regulation)

protein destruction

• marked for destruction by the addition of ubiquitin on exposed lysine residues

most common PTM types

1. phosphorylation

2. glycosylation

3. acylation

4. alkylation

5. hydroxylation

phosphorylation

• phosphate group added to certain amino acid

  • serine (ser), threonine (Thr), Tyrosine (Tyr)

glycosylation

• sugar moieties attachment to nitrogen/oxygen atoms in side chains of amino aids

  • Aspargine, Serine, Threonine

Acylation

• acyl group linked to the side chain of amino acids

• aspargine, glutamine, lysine

alkylation

• alkyl group (eg. methyl grp) added to amino acids

• lysine or arginine

• longer chain alkyl grps may also attach in some cases

hydroxylation

• PTM mostly found on proline and lysine residues which make up the collagen tissue

• enables crosslinking which strengthens muscle fibers

protein synthesis

1. PTM

2. Protein translation

3. Cytosol

4. Signal sequence

5. Endoplasmic reticulum

6. Cleaved protein

proteins

• polymers of amino acids joined together by peptide bonds

• final products of gene expression

protein classification (CSSR)

by CHARGE

• anionic (negative) - 95-97%

• cationic (positive) - 3-5%

by SHAPE

• globular - ball-like/compact for functional

• fibrous - long, strand-like for structural

by SIZE

• small, medium, large

  • based on amino acid length

  • based on molecular weight

by ROLE/FUNCTION

• metabolic proteins

• structural proteins

• regulatory proteins

amino acid structure

peptide bond formation

-OH release to connect to the amino group of another amino acid

Chemical Families of Side Chains

1. nonpolar, aliphatic

2. polar, uncharged

3. positively charged

4. negatively charged

5. nonpolar, aromatic

nonpolar, aliphatic (hates water)

1. glycine

2. alanine

3. valine

4. leucine

5. methionine

6. isoleucine

polar, uncharged

1. serine

2. threonine

3. cysteine

4. proline

5. asparagine

6. glutamine

positively charged

1. lysine

2. arginine

3. histidine

negatively charged

1. aspartate

2. glutamate

nonpolar, aromatic

1. phenylalanine

2. tyrosine

3. tryptophan

Structures of proteins

amino acid sequence determines how a protein folds, and the final shape determines what the protein can do

1. Primary

2. Secondary

3. Tertiary

4. Quaternary

primary protein

PROTEIN FOLDING

• exact sequence of amino acids in a polypeptide

  • had diff R-group to interact w/ other R groups, water, and protein backbone

Primary structure → determines folding → determines 3D shape → determines function

secondary structure

• local spatial conformation of the polypeptide backbone

• two common structural elements (repetitive)

  • α-helix

    • backbone coils like a spring

  • β-sheet

    • backbone folds back and forth, creating a sheet-like structure

• holds together by hydrogen bonds between peptide backbone

beta turn (non-repetitive structural)

• 4 residues

  • Carbonyl O of residue n «» N-H of residue n+3

• short structure that reverses the direction of the polypeptide chain

omega loops

• flexible region that connects structured regions such as α-helices and β-sheets

α-helix → loop → β-sheet → loop → α-helix

tertiary structure

• three-dimensional structure of a single polypeptide chain

• R-group interactions

  • charges - attractions help stabilize the protein's structure

  • hydrophobic - inside protein

  • hydrophilic - outside protein

  • disulfide bonds - Cys – S – S – Cys

    • (some amino contain sulfure)

    • relatively stronger due to covalent bonds

    • more resistant to denaturation (structure and biological activty)

quaternary structure

• TWO OR MORE polypeptide chains

• individual chains are called: Subunits

Recombinant DNA

• DNA made by combining genetic material from two different sources

Polymerase Chain Reaction

• amplification

• increase the number of copies of a particular DNA sequence

Plasmid

• acts as a vector that carries foreign genetic material into a host cell

Restriction endonuclease

• cuts the sugar-phosphate backbones

sticky ends

• produced when annealing the fragment to the plasmid when both molecules are cut by the same restriction enzyme

DNA ligase

seals the sugar-phosphate backbone of DNA fragment that have been joined

Transformation

• plasmid to bacteria

• Electroporation

• Heat shock

Electroporation

• bacteria exposed to a sudden change in temperature create temporary openings in the bacterial membrane

Heat shock (42C)

• short electric pulse create temporary openings in the bacterial membrane

Blue-white screening

• Selection of bacteria with transformants

• Blue - plasmid w/o inserted gene

• White - plasmid w/ inserted gene

Ampicillin

• kills the cells that lack the inserted gene

Antibiotic resistance gene

• helps identify bacteria that successfully received the plasmid

Tissue-specific promoter sequence

• controls where the transgene is expressed (eg. leaves, roots, fruits)

Transgene (gene of interest)

• desired gene inserted to produce a specific trait or function

stop sequence

• signals the end of gene transcription

• telling to stop copying the gene into RNA

Techniques to Insert Genes in Plants

1. Particle Bombardment or Biolistics (Gene Gun)

2. Agrobacterium-mediated

Particle Bombardment or Biolistics (Gene Gun)

• uses gene construct/naked DNA

  • coated onto very small metal particles, commonly gold or tungsten

• Microparticle Bombardment Biolistics and Gene Gun

  • use of high-velocity microprojectiles to penetrate the out/inner layer of the plant cell

DNA → coat tiny particles → shoot → penetrate → DNA enters cell → gene expression

Agrobacterium-mediated

Agrobacterium tumefaciens

• naturally transfer a piece of its DNA into a plant cell

  • Ti (tumor-inducing) plasmid

    • allows Agrobacterium to cause tumor formation in infected plants

  • T-DNA in Ti plasmid

    • fragment transferred from the bacterium into the plant cell

    • left and right border tells what DNA region should be transferred

      • RB - T-DNA transfer starts

      • LB - end of transfer

Binary Vector system

• introduce foreign DNA to agrobacterium cells

  • created from Ti plasmid but with modification instead of having one huge Ti plasmid containing everything, the functions are separated into two plasmids

Modern Biotechnology

1. Polymerase chain reaction (PCR)

2. Electrophoresis

3. Sothern, Northern, and Western Blotting