Post Quiz 4 Content

lec 45 - end

Drosophila developmental cycle

oocyte → zygote → early embryo- no segments → late embryo (segmented) → larval station → pupa → adult

embryonic development stages

• 1 oocyte with many nurse cells

• oocyte exbands, retrackting the nurse cells as follicle cells develop

• mature oocyte can be fertilized (becomes a zygote)

• rapid nuclear division in 1 cell

• membrane begins to invaginate

  • forms a cellular blastoderm

Bicoid - maternal gene product

• biocoid mRNA is synthesized by nurse cells and deposited in the unfertilized egg near its anterior pole

• after fertilization, biocoid mRNA is translated to make Bicoid, acting as an anterior TF

• makes a concentration gradient from anterior to posterior

5 main TF cascades that drive cell fates

1. bicoid (anterior → posterior)

2. kruppel & hunchback (gap genes)

3. Eve & ftz (pair-rule gene)

4. Caudal (posterior → anterior)

5. Nanos (posterior → anterior)

   1. both bicoid and nanos comes from the mother laying mRNA

6. the opossing TF gradients + cooperative binding sharpen transitions of gene expression!

Segmentation Genes

direct the formation of the proper number of body segments

• transcribed after fertilization

gap genes

• divide the developing embryo into several b road regions

• ex: kruppel and hunchback

pair-rule genes + segment polarity genes

define 14 stripes that become the 14 segments of an embryo

• ex: Eve and Ftz

Homeotic genes

Specify which organs/appendages will develop in particular body segments

• typically encode TFs that contain a homeodomain

• encode Hox genes

Homeodomain

• bind specific DNA enhancer sequences

Hox gene clusters

responsible for the development of structures in a defined part of the body

• Drosophila have 1 Hox gene

• humans have 4!

There is a lot of conservation between Hox and mammalian hox complexes!

stem cells

cells that can differentiate into various tissues

2 main functions!

1. replenishing themselves

2. providing cells that can differentiate

totipotent cells

cells that can differentiate into ANY TISSUE or a complete organism

pluripotent cells

can give rise to cells of all 3 germ layers as well as many tissue types

• cannot differentiate into a complete organism

embryonic stem cells

pluripotent cells of the blastocyst

unipotent cells

can develop into only 1 type of cell/tissue

adult stem cells

• more limited potential than embryonic stem cells

  • considered multipotent

  • have a niche: microenvironment that promotes stem cell maintenance while allowing differentiation of some daughter cells

niche

microenvironment that promotes stem cell maintenance while allowing differentiation of some daughter cells

• receives signals from neighboring cells to maintain the stem cell lineage

signaling of neighboring cells

• can cause a cell to commit to differentiation or die through apoptosis

codon

triplet of nucleotides that codes for a specific amino acid with specificity

How are codons degenerate?

There are 4 code letters and 3 spots within a codon (4³=64) however there are only 20 AA

• multiple different codes encode for 1 AA however, the 1 code NEVER codes for multiple AAs.

specificity of reading frames

• open reading frames have a start codon that have the longest sequence without a stop codon.

• evolved to remove sequences that could prematurely stop UNLESS there is a frameshift

  • then it would likely encounter a premature stop codon, forming a truncated protein

How were AA codon codes found?

• poly(u) and 20 radioactive amino acids were fed to E.Coli and it found that UUU codes for Phe

  • same approach with CCC for proline, AAA for lysine

The 3 termination codons

UAA, UAG, UGA

• also called nonsense codons

• do not code for any AA

Open reading Frame

• starts with AUG (Met), initiation codon, followed by at least 50 codons, and ends with a stop codon

anticodon

A 3-base sequence on the tRNA that base pairs with mRNA

• pairing occurs via hydrogen bonding

• it is an antiparallel alignment

• Only 32 tRNAs (not 61) are needed, due to the wobble effect

wobble effect

• the third codon is a degenerate psotition

• much weaker H-bonding

• if there is a mutation at the 3rd position, mutations are likely to be silent (encode for the same AA)

Resiliency in code mutation

• Mutations of a codon usually produce a conservative substitution

• ex: valine → alanine

  • both are nonpolar!

missense vs. nonsense

missence - mutations that change the encoded amino acid

nonsense - mutations that make a stop codon

Indels

• insertion or deletion. Only indels with a length the multiple of 3 will maintain the reading frame.

• when out of frame, a stop codon is likely to be encountered

encoded translational frameshifting

aka “hiccupping” of ribosomes

• allows 2+ related but distinct proteins to be produced from 1 transcript

• occurs during translational frameshifting

• 2+ related but distinct proteins can be produced from 1 transcript

Five stages of protein synthesis

1) activation of amino acids (when tRNA is aminacylated)

2)Initiation: the mRNA and the aminoacylated tRNA bind to the small ribosmal subunit, then the large ribosomal subunit binds

3) Elongation: successive cycles of aminoacy-tRNA binding and peptide bond formation occur until the ribosome reaches a stop codon

4) Termination: translation stops when a stop codon is encountered. The mRNA and protein dissociate, and the ribosomal subunits are recycled

5) Protein folding: posttranslational processing

essential components for the activation of amino acids in E. Coli

• all 20 AA, 20 aminoacyl-tRNA synthases and 32 or more tRNAs

• ATP and Mg2+

Step1 for AA activation

• tRNAs are charged when attaching their amino acid (aminoacylated)

• aminoacyl-tRNA synthetases esterify the 20 AA to their corresponding tRNAs

• each is specific for 1 amino acid and 1 or more corresponding tRNAs

• occurs in the cytosol

• ATP activates the carboxyl group of each AA

Step 2 for aminoacyl-tRNA synthetases

• attach the correct amin oacid to their tRNA as defined by their anticodon

• transfer the aminoacyl group from teh enzyme0bound aminoacyl-AMP to the corresponding specific tRNA

• The aminoacyl group is esterified to the 3’ position of the terminal A m=nucletodide of the tRNA

• the ester linkage activates the AA and joins it to the tRNA

More on Aminoacyl-tRNA

• aminoacyl-tRNA synthetases must match

Essential components for protein synthesis initiation in E. Coli

• mRNA

• fMet in prokaryotes

• Initiationcodon (AUG)

• 50S ribosomal subunit

• Initiation factors (IF1,2,3)

• GTP

• Mg2+

Essential components for protein synthesis elongation in E. Coli

• functional 70S ribosomes (initiation complex)

• aminoacyl0tRNAs specified by codons

• elongation factors (EF-Tu, EF-Ts, EF-G)

• GTP

• Mg2+

Essential components of termination and ribosome recycling in E. Coli

termination codon in mRNA

Release factors (RF1,2,3, RRF)

EF-G

IF3

Folding and posttranslational processing

• chaperones and folding enzymes (PPI, PDI)

• specific enzymes, cofactors, and other components for the removal of initiating residues and signal sequences

• modifications of terminal residues

• attachments of acetyl, phosphoryl, methyl, carboxyl, carbohydrate, or prosthetic groups

tRNA structure

• AA arm - carries a specific amino acid esterified by its carboxyl group to the 2’-OH or 3’-OH group of the A residue at the 3’ end of the tRNA

  • emphasis on esterified AA!

• anticodon arm

  • contains the anticodon

Crick’s adaptor hypothesis

• A small nucleic acid could act as an adaptor, binding to both a specific AA and the mRNA econding that AA

• amino acids are activated for protein synthesis

  • aminoacyl-tRNAs: tRNA attached to an amino aicd

  • aminoacyl-tRNA synthetases

    • catalyzed the formation of aminoacyl-tRNAs