cells exam ii

conn coll!

Reporter genes

ex. GFP (glow), luciferase (glow0, Lac Z (blue). They are used by scientists to identify certain segments of a gene and seeing if they get expressed. Replace target gene, such as FXR, and when it gets expressed, it glows.Reporter genes are genetic markers that produce measurable signals, like fluorescence or color, indicating the expression of a target gene. They help in studying gene regulation and the functionality of gene sequences.

What methods used to levels of transcripts?

Real Time PCR:

also uses reverse transcription polymerase reaction ; number of input DNA increases, the time to detect fluorescence, decreases.

—> transcript levels in different tissues

and Reverse Transcriptase PCR:

isolate desire mRNAm, TTTT primers to poly AAA tail, use reverse transcriptase to copy RNA into DNA, separate strands + second primer, now use PCR to amplify strand + use primers from gene being studied

Post Transcriptional Controls

• Alternative splicing: allows one single gene to make multiple mRNAs and therefore proteins. Regulated by RNA binding proteins → how transcript will be spliced → can have very different results

• mRNA localization and translation

• RNA binding proteins

  • RNA binding proteins represses translation of its own RNA when found in excess

  • also high temp

• miRNAs direct destruction of target mRNAs, regulate gene expression

  • these partially double stranded RNA Molecules first bind to Dicer (not shown) that cuts the ds RNA into ~21 bp pieces. The dsRNA then binds to proteins to form the RISC (RNA-induced Silencing Complex). One of the two strands of RNA are eliminated. . The remaining RISC searches for complementary mRNA sequences and either destroys them or inhibits their translation.

• siRNAs DO NOT regulate gene expression - shut off viruses and function similarly

You identify a protein, named Camel, that affects the expression of the gene named Conn. Upon further investigation, you determine that the Camel protein sequence includes a section that is found in many RNA binding proteins. Name one way that the Camel protein might be affecting the expression of Conn based on these findings

1. The Camel protein could bind the Conn mRNA to affect it’s splicing such that the Conn mRNA is spliced differently in different tissues.

2. The Camel protein could bind the Conn mRNA to transport it for specific cellular localization.

3. The Camel protein could bind the Conn mRNA preventing it from being translated into a protein.

Protein Structure

• Primary: amino acids with R groups linked together by peptide bonds (strong covalent)

  • R groups can be charged or uncharged, polar or nonpolar

  • 3 letters, 20 in total - codon

  • determine structure and function

• Secondary

  • alpha helices and beta sheets

  • held together by backbone to backbone H bonds (both beta and alpha)

  • both can be in one protein → form tertiary

    • H bond bonded to O of C=O bond in backbone

  • how membrane bound proteins cross the lipid as alpha helix

  • beta sheets can be parallel or antiparallel

  • first described!

• Tertiary:

  • 3D structure of entire polypeptide chain

• Quaternary:

  • 3D structure of multiple polypeptide chains

  • ex. neuraminadase is 4 polypeptides

  • symmetrical assembly of 2 different polypeptides

N terminus: amino

C terminus: carboxyl

• polypeptide backbone does not include R chains!!

Bonds that help proteins fold?

Covalent (peptide bonds) initial structure and then: hydrogen bonding,Van der Waals, electrostatic attractions, hydrophobic forces, disulfide bonds (covalent) form between two cysteine form keratin → stabilize 3D structure all together

Protein folding

• denatured proteins can offer recover shape

  • all info for 3D is in primary structure

  • ex. urea/denature remove/reform

• Chaperone proteins

  • assist in protein folding

    • some bind to partly folded chains and help them form

    • others form an isolation chamber in which single polypeptide chains can fold correctly

Why is protein folding important?

• improper folding leads to disease, such as prion diseases (neurodegenerative diseases)

  • Madcow/Creutzfeldt-Jakob disease

How do proteins work?

Ligands are any substance bound to a protein

• the ability of a protein to bind selectivley to a ligand is due to the 4 noncovalent interactions

  • van der waals

  • hydrogen bonds

  • electrostatic attractions

  • hydrophobic forces

• region of protein for ligand: binding site

  • allows proteins to interact with specific ligands - like cyclic AMP

    • made of particular amino acid (R) chains

Enzymes

• enzymes are proteins but not all proteins are enzymes

• even energetically favorable reactions require activation energy to get started → ex. heat or enzymes

• enzymes lower the activation energy of a reaction → can speed up rxns

  • a series of enzyme catalyzed reactions can create a linked pathway

• energetically favorable - Δ G

• energetically unfavorable + Δ G

  • reaction coupling to push unfavorable rxn

• Enzymes convert substrates to products while remaining unchanged

How do enzymes lower activation energy?

• Holding reactant substrates together in precise alignment to encourage rxn

• binding of substrate to rearrange electrons in the substrate creating partial + or - to favor reaction

• enzyme strains the bound substrate forcing to a transition state that favors rxn

• a single enzyme might use a combination of these mechanisms

• ex: lysozyme

  • Lysozyme lowers the activation energy by:

  • 1. Straining the substrate

  • 2. Altering the distribution of electrons in the substrate

  • hydrolysis +h2O rxn

• binding site is referred to as the active site

Vmax

maximum rate that an enzyme can convert a substrate to a product

Km

How well the enzyme binds to substrate

assay

way to measure the disappearance of a substrate and appearance of product

ex. using spectrophotometer

competitive inhibitor

blocks substrate from binding to desired enzyme, increases Km but Vmax stays the same

ex. Gleevec

What is the purpose of enzymes?

Enzymes speed up reactions by lowering the activation energy

Enzymes drive biological reactions in cells without the cells having to raise their temperature Enzymes usually catalyze the formation or breakage of a single bond

Enzymes bind to a substrate in their active site

Enzymes and substrates have a characteristic Vmax and Km that can be determined experimentally

Transcription Regulators

• 3 alpha helices of transcription regulator bind to sequence of DNA → homeodomain

• sometimes ligand is DNA → these specific interactions with amino acids and DNA molecules demonstrate how it can bind to specific enhancers (DNA sequence)

How are proteins controlled?

• allosteric proteins have two or more binding sites - saw with trp operon

• feedback inhibitions: an enzyme that catalyzes an important reaction is turned off when a substance is present in high amounts

• phosphorylation (covalent bonds): causes a conformational change which can either decrease or increase a proteins activity by adding a phosphate group

• Groups that are covalently attached: multiple sites can be modified by other phosphates, acetyl (histones), fatty acids, sugars, and ubiquitin - can change conformation and mass

• Non covalent GTP binding - molecular switches

Kinase: Enzyme that catalyzes the transfer of a phosphate group from ATP to a molecule

Phosphatase: Enzyme that catalyzes the removal of phosphate from a molecule