CELL 201: Lecture 5 - Protein Synthesis + Sorting

****What does the S in different Ribosomes (eg. 16S rRNA) stand for?

Svedberg unit = has to do with SEDIMENTATION RATE

****Define Sedimentation Rate

Rate of movement through a solution when an object is subject to centrifugal force

***What does the Sedimentation rate depend on?

SIZE, shape + DENSITY

****What is Differential Centrifugation?

Technique that takes advantage of Sedimentation coefficient (svedberg unit) to separate different particles based on SIZE + DENSITY

****How do you do Differential Centrifugation/ how does it work?

• Start with homogenization

  • Break open cells → creates a cell homogenate.

• First spin (low speed)

  • The heaviest things (like nuclei) pellet at the bottom.

  • The lighter stuff stays in the liquid (supernatant).

• Second spin (higher speed)

  • Take the supernatant and spin faster.

  • Next-heaviest structures (like mitochondria, lysosomes, peroxisomes) pellet.

• Keep spinning faster

****Can all cell parts be separated by Sedimentation Coefficient + centrifuge?

NO!

• Some organelles have same Sedimentation coefficient = can’t be separated by centrifuge

****If centrifuge won’t work for those with same sedimentation rate WHAT should be done instead?

Density gradient centrifugation

****How does Density gradient Centrifugation differ from Differential Centrifugation?

Separates particles by their buoyant density (not just size).

• can distinguish molecules of similar size but different densities.

****What is the mechanism behind Density gradient Centrifugation? Aka how does it work?

Sample is layered on a gradient of solutions. After centrifugation:

• Each particle moves until it reaches the spot in the gradient where its density = the gradient’s density.

• Components form distinct bands, not just pellets.

*****define SUBCELLULAR FRACTIONATION

The process of using centrifugation to Isolate + purify organelles + macros based on sedimentation rates + density

Are prokaryotic cells monocistronic?

NO they are poly cistronic

EUKARYOTES = monocistronic (1 mRNA = 1 protein)

What are the Polycistronic transcription units of Prokaryotes called?

OPERONS

****What are the 5 machineries involved in translation? What do they each do?

1. Ribosome = polypep synth.

2. mRNA = encodes polypep. sequence

3. Aminoacyl-tRNA synthases = Attach aa to their appropriate tRNA

4. tRNA = deliver aa

5. Regulatory protein factors = facilitate initiation, elongation + termination of translation

***WHERE are ribosomes found in Eukaryotes? (2 places)

• Free in the cytoplasm

• Bound to Rough ER + outer nuclear envelope

****Where is the mRNA binding site located on the ribosome?

SMALL subunit

****What are the 3 sites for poly-pep synthesis in the Ribosome + what do they each do?

A = Where tRNA brings the aa

P = where the polypep. chain grows

E = Where the empty tRNA exits

*****How are Amino Acids linked to the tRNA? What type of BOND?

ESTER BOND

****WHERE is this ester bond located on the tRNA? 3’ or 5’?

3’ End

***There are over 500 tRNA genes in the human genome. WHY do we have so many when we only need 61?

Redundancy allows us to loose a few tRNAs in the instance of  a mutation to prevent future issues with translation (which would effect all proteins)

****What is an AMINOACYL tRNA?

tRNA attached to an amino acid

*****What does it mean for the tRNA to be CHARGED?

It has an aa attached

*****When is an aa ACTIVATED?

When it is attached to a tRNA

****How does the tRNA recognize the codon in mRNA?

complementation of the ANTICODON

****What direction is mRNA read and why?

read 5’ — 3’ because mRNA = synthesized 5’ to 3’ + can be read as soon as possible

*****What is an AMINOACYL-tRNA SYNTHETASE?

the thing that LINKS aa to tRNA

****How many different aminoacyl-tRNA synthetases do cells have?

20

• one for each aa

*****What are the 4 STEPS involved in making the aminoacyl-tRNA?

1. aa + ATP enter synthetase

2. AMP attaches to aa + releases a pyrophosphate

3. AMP is displaced by tRNA creating an aminoacyl-tRNA'

4. Aminoacyl-tRNA = released

***The aminoacyl-tRNA synthetase must recognize BOTH the amino acid + the tRNA. HOW does it do each?

aa: recognize by chemistry + shape

tRNA: all have the same shape

• recognizes the ANTICODON

When synthesizing the polypeptide, is it the N-terminal that is synthesized first or the C terminal?

N-terminus

***What are the 3 stages of Translation?

1. Initiation

2. Elongation

3. Termination