Module 3: The Endoplasmic Reticulum

Section 1:

 Synthesizes proteins and lipids

 Transports molecule around the cell
 Made of cisternae
 Cisternae connected to outer membrane of nuclear envelope

Rough Endoplasmic Reticulum:

 Studded with ribosomes
 Rough appearance under electron microscope
 Ribosomes are made up of rRNA and ribosomal proteins
 They are enzymes responsible for translation
 Assemble at the RER when proteins produced need to be transported to other
organelles (endomembrane system)
 Ribosomes present in the cytosol, freely floating
 Proteins produced by these ribosomes stay in the cytosol

Smooth Endoplasmic Reticulum:

 No ribosomes on surface
 Smooth when viewed under an electron microscope
 Produce lipids, phospholipids and steroids
 Carbohydrate metabolism
 Convert glucose-6-phosphate into glucose
 In muscle cells, control calcium ion concentration-important for muscle contraction

Section 2: RNA Review

mRNA:
 Translated into proteins
 Coding template for peptides

tRNA:
 Linker molecule
 Links amino acids to the mRNA
 Smaller in size-73-93 nucleotides
 Non-coding

rRNA:
 Make up the ribosome complex to translate mRNA into proteins
 2 subunits
 Large subunit-5000 nucleotides
 Small subunit-1900 nucleotides
 Non-coding

siRNA:
  Binds to mRNA
 Degrades and turns off gene
 Non-coding

Section 3: The Central Dogma Part 2 (Transcription)

Important Proteins in Transcription:

RNA Polymerase:
 Enzyme
 Synthesizes RNA from DNA
 RNA Polymerase I-rRNA
 RNA Polymerase II (pol II)-mRNA
 RNA Polymerase III-tRNA

Transcription Factors:
 Not all genes are transcribed in a cell at the same time
 Transcription factors (proteins) bind to the regulatory sequence of the gene
 Signal the transcription machinery about which gene needs to be transcribed
 Work independently or with other proteins
 Control rate of transcription from DNA to mRNA
 Able to promote (activator) or block (repressor) the transcription of genes by altering
ability of RNA Polymerase to start transcription

3 stages of Transcription:

Initiation:

 Starting step of Initiation

 Transcription factors bind to the Transcription Start Point
 Upstream of the TSP, there are promoters
 Promotors closest to the TSP is the core promotor
 The TF binds to the TATA box on the core promotor
 Once the TF are bound to the TATA box, the RNA Polymerase II can bind to the
promotor
 TF binding to the TATA box does three things:
 Guides RNA Polymerase II to the correct DNA strand
 Activates RNA Polymerase II by adding 2 phosphate groups (phosphorylation)
 Unwinding the DNA strands enough for RNA Polymerase II to access gene being
transcribed (exposes template strand)
 RNA Polymerase synthesizes first few nucleotides of RNA molecule while remaining
stationary at the promotor.
Elongation:

 The RNA Polymerase II extends the RNA molecule 5’-3’ while reading DNA template
strand (synthesizes the remainder of the RNA molecule)
 The unwound DNA (transcription bubble) is covered by RNA Polymerase II to protect
it from damage

Termination:

 There aren’t proper methods to stop transcription

 Proteins can bind to RNA Polymerase II
 RNA is separated from RNA Polymerase II by an enzyme
 RNA Polymerase does not have proofreading function and therefore lower fidelity
than DNA Polymerase
 RNA Polymerase synthesizes short molecules of RNA until one has proper
complimentary hydrogen bonds with the complimentary DNA strand
 Ensures correct mRNA molecule being made
 This is when RNA Polymerase reaches a location on the template strand where no
additional ribonucleotides are added to the RNA
 Transcription Bubble collapses
 RNA molecule dissociates off template
 RNA Polymerase falls of DNA

DNA Replication RNA Transcription

Nucleus Nucleus
Replication of cell’s entire DNA mRNA made from one or few genes
In both directions Single direction (5’-3’)
Okazaki Fragments Tightly regulated areas of genes
All DNA is replicated
Proofreading function Short molecules of RNA are synthesized
DNA repair occurs constantly until one has proper complimentary
hydrogen bonds with the DNA strand
Not proofread
Replication occurs in preparation for cell Cell and time specific
division Gene specific promotors and TF
DNA Polymerase RNA Polymerase II

Post-Transcriptional Modification:

 RNA is short lived in the cell

 Cell’s needs are changing-different proteins required at different times
 However, the mRNA needs to be kept around long enough that the protein the cell
needs

5’ Methyl Guanosine Cap:

 Guanosine Triphosphate is added to the 5’ carbon of the mRNA molecule

  Via a 5’-5’ triphosphate linkage
 Immediately after capping, the GTP becomes methylated
 Methyl group on 7’ of guanosine base
 Methyl guanosine cap present on all eukaryotic mRNA
 Protects mRNA molecule from premature degradation by enzymes in the cell that
degrade nucleotides and nucleases

3’ Polyadenylation:

 Occurs after 5’ Methyl Guanosine Cap

 After mRNA is cut off from RNA Polymerase II
 A new Polymerase attaches 200 adenosines to the 3’ Carbon of mRNA
 Forms a Poly(A)tail
 Necessary for binding protein that are needed to transport the mRNA outside the
nucleus to start translation

RNA Splicing:

 Exons: Coding regions

 Introns: Non-coding regions (not part of mature proteins)
 Introns need to be removed from the RNA as they do not code for proteins
 Some genes use alternate splicing
 Multiple mRNA molecules produced from a single gene-exons skipped
 2 mRNA molecules produced at least
 Mutations at splicing sites-incorrect splicing-improper protein translation

Transport through NPCs:

 Proteins bind to mRNA

 Transport mRNA into the cytosol
 Some proteins recycled and transported back to the nucleus
 Some assist mRNA in translation
 mRNA remains in cytosol

Section 4: The Central Dogma Part 3 (Translation)

 mRNA is translated to proteins (RNA Codons to amino acids)

 4 DNA bases – 20 amino acids
 Creates sequence of 3 RNA nucleotides (codon)
 Codes for an amino acid/stop signal during protein synthesis
 3 nucleotides long- 64 possible combinations
 Enough for 20 amino acids and a stop codon
 1 codon-1 amino acid
 Multiple codons can be for the same amino acid
 Most variation in the third position of the codon (e.g. Glycine)

Components of Translation:
Small ribosomal subunits:
 mRNA binds

Large ribosomal subunit:

 A: tRNA attaches
 P: Newly arrived amino acid is removed from its tRNA and added to the peptide
chain via peptide bonds
 E: tRNA is ejected from the ribosome
 Polypeptide Exit Tunnel: Peptide is guided and released

Initiation Factors:
 Bind to mRNA
 5’ Methyl Guanosine cap
 Helps the small ribosomal unit to identify the initiation site (Small ribosomal subunit)

Elongation Factors:
 Assist in elongation.
 Form complexes to deliver tRNA and GTP source to ribosome (large ribosomal
subunit)

tRNA:
 Recognizes the codon
 Is attached to accompanying amino acid
 Delivers correct amino acid to peptide chain
 Has an anticodon
 Anticodon is complimentary to codon
 Bond between tRNA and amino acid provides energy to make a new peptide bond
on the growing amino acid chain

mRNA:
 Protein factors from the nucleus deliver mRNA to the cytosol
 Depending on its starting sequence, it stays in the cytosol or is transported to the ER

Translation Steps:

Initiation:
 The small ribosomal subunit attaches to the mRNA molecule near the 5’ MG cap
where initiation factors are bound
 Crawls forward until start codon (AUG) is found
 Initiator tRNA binds to this
 Large ribosomal subunit encloses the mRNA
 The initiator tRNA is in the P position

Elongation:
 Multi-step cycle
 Continuous loop
  Each cycle adds an amino acid to the growing peptide chain
 Energy expending process
 Energy rich molecules such as GTP are added in each step
 Previously used tRNA is ejected from the E site and new tRNA binds to the A site
 Peptidyl Transferase (enzyme in large ribosomal subunit) moves growing peptide
chain from the P site to the A site so it can attach to the amino acid on the tRNA
 Ribosome translocate down one codon on the mRNA at a time
 mRNA and tRNA go from A and P sites to P and E sites

Termination:
 Stop codons: UAA, UGA, UAG
 Not connected to tRNA molecules
 Attract release factors
 Release factors occupy A site
 Add water instead of amino acid to the peptide chain at the P site
 Forms a carboxylic acid
 Peptide is released
 Then, ribosome release factors bind to the A site
 Release of mRNA from ribosome
 mRNA is recycled for more translation

Mutations:
 Changes in DNA structure that result in variant form that can be passed onto
daughter cells
 Changes in DNA directly change RNA
 Can affect amino acid coded for depending on how codons are read
 Can result in a mutated protein
 Positive: species evolve and proteins improve function
 Negative: illnesses and diseases

Types of Mutations:

Point Mutation:

 Single nucleotide is changed

 Could be Silent, Missense or Nonsense
 Silent: Amino acid does not change
 Missense: Amino acid changes
 Nonsense: Amino acid codon is replaced with a stop codon which stops translation
and prevents the production of the rest of the amino acid which is detrimental-
especially if a mutation occurs near the start of a sequence

Insertion:

 Extra base pair is added to sequence

 Shifts the 3-base pair reading frame down by 1
  Alters every amino acid
 Similar effect with 2 base pairs added
 With 3 base pairs added, a new amino acid is added

Deletion:

 Base pair is removed

 Alters reading frame
 Same effect is Insertion unless is multiples of 3

Large scale deletion, insertion and recombination:

 Involve entire chromosomes or parts of chromosomes

 Lethal

Section 5: Amino Acids and Proteins

 Amino group
 Alpha carbon with R group
 Carboxylic acid group

Types of Amino Acids:

 Depends on properties of R group

Hydrophobic Amino Acids:

 Nonpolar
 Aliphatic
 R group is a straight carbon chain or aromatic
 Found in the core of the protein or interacting with other hydrophobic molecules
(fats or lipids in the membrane)
 GAVLIMP
 Glycine (no R group-smallest-flexible)
 Alanine (single carbon methyl group-small)
 Valine
 Leucine
 Isoleucine
 Methionine
 Proline (rigid-end of side chain forms covalent bond with nitrogen of amino group)

Aromatic Amino Acids:

 Ring structures with double bonds
 Large
 Gain or loss of these amino acids can result in deformities in the protein
 Tire tripped on Phenyl
 Tyrosine (Polar)
  Tryptophan (NP)
 Phenylalanine (NP)

Hydrophilic Amino Acids:

 Polar
 Side chains can form hydrogen bonds that stabilize proteins
 Found on the outside of a protein
 Serena had a three-year-old tire that she ate with asparagus, Glucose and Cysts
 Serine
 Threonine
 Tyrosine (Aromatic)
 Asparagine
 Glutamine
 Cysteine (sulfate that forms disulfide bonds with another Cysteine-significant in
forming 3D protein structure)

Charged Hydrophilic Amino Acids:

 Outside of protein
 Interact with water
 Positive: Lysol in Argentinian History
 Lysine
 Arginine
 Histidine
 Negative: Aspartic Acid, Glutamic Acid

Peptide Bonds:

 Links amino acids

 Carboxylic group of one amino acid and amino group of another amino acid
 Dehydration reaction
 Not between R groups
 Amino acids can rotate about the peptide bond
 Amino Terminus
 Carboxy terminus

Primary Protein Structure:

 Linear peptide sequence-sequence of amino acids
 3 letter codons or single letter amino acid codes
 N Terminus to C Terminus numbering
Secondary Protein Structure:
 Regions of organization in peptide sequence
 Alpha helix: tight coil that forms hydrogen bonds with the backbone of every 4 th
amino acid
 Beta Sheets: planes are formed between rows of amino acids with hydrogen bonds
between the backbones
 Parallel or antiparallel
Tertiary protein Structure:
 3D configuration of the protein
 Defined by secondary structure and domains of protein
 To properly fold, molecular chaperones need to be present (other proteins)
 Disulfide bonds can be present

Quaternary Structure:
 Complex of multiple proteins
 Individual proteins are called subunits if they cannot function independently outside
the complex

Domains:

 Discrete structural unit that is assumed to fold independently of the rest of the
protein and to have its own function
 Certain protein domains like enzymes have some clearly associated function with
them
 Such domains have the same function when they get inserted into different proteins
during evolution
 20-100 amino acids
 Multiple secondary structures
 Most proteins are multi domain and are conserved between evolutionary related
proteins
 Zinc Fingers, transmembrane, SH2 or are named alphabetically (domain A, B, C)
 Yellow protein: SH3 domain
 Purple Protein: LIM domain
 Green protein: Kinase domain

Protein Shape and Function:

 Proteins have different conformations depending on environmental conditions

 This alters how they function and interact with other proteins
 pH
 Temperature
 Presence of ions such as Mg or Ca (alter folding)
 Structure and function are directly related (Lock and Key Hypothesis)

Protein Modification:

 Long lasting: Covalent modifications

 Disulfide bonds
 Addition of sugar structures
 Phosphate groups, Methyl groups or Acetyl groups
 Activate or inactivate proteins
 Change how they interact with other proteins
 Short-lived: Non-covalent modifications
 Proteins interacting with each other in binding sites or small molecules like Ca or Mg
binding transiently