Cell Bio Review

a nucleotide is to a nucleic acid

DNA

are not true polymers

have double bonds in their fatty acid chains

CH4

added; dehydration synthesis

polysaccharide

glycogen

enzymes; proteins

R group

nRNA

a codon

C.

codons code for trinucleotides, amino acids are not encoded by codons, codon and anti are different

transcription

promoter

translation

ATG

D

C

B

molecules form by removing water

molecules bread down by adding water

sequence of its amino acids

formed by hydrogen bonds between nonadjacent carboxyl and amino groups

results from interactions between R-groups

results from hydrogen and ionic bonds between separate tertiary structures

solid crystals of purified protein are placed in an X-ray beam, and the pattern of deflected X rays is used to predict the positions of the atoms within the protein crystal

adenin, guanine, cytosine, thymine (DNA), uracil (RNA)

OH or H in sugar base

U or T

single strange or double strand

RNA transfers the genetic information from DNA to the protein-making machinery

DNA stores the genetic information

two hydrogen bond

three hydrogen bonds

has a C,H,O usually 2:1 (Hydrogen:Oxygen)

not water soluble, made of fatty acid chains that store energy

fat → triglyceride

each carbon in the hydrocarbon chain is bound to two hydrogen atoms

tightly packed, solid at room temp

at least one carbon in the hydrocarbon chain is bound to just one hydrogen

loosely packed, liquid at room temp]

replication → DNA → transcription → RNA → translation → protein

process by which DNA makes a copy of itself during cell division

1. replication fork formation

2. primer binding

3. elongation

4. termination

process of DNA replication where it is unzipped into two single strands via DNA helices, an enzyme that disrupts the hydrogen bonding between base pairs

only processes in the 5’ to 3’ direction

5’ has a phosphate group attached and 3’ has a hydroxyl group attached

but, it is bidirectional so 3’ to 5’ is the leading strand and 5’ to 3’ is the lagging strand.

in DNA replication, a short piece of RNA that will bind to the 3’ end of the strand (the starting point for replication)

generated by the enzyme DNA primase

process during DNA replication where DNA polymerases will create the new strand

DNA pol III is the main replication enzyme while DNA polymerases I, II, IV, V, are responsible for error checking and repair (prokaryotic cells)

In eukaryotic cells, there are DNA pol alpha, delta, epsilon

replication proceeds in the 5’ to 3’ direction on the leading strand, the newly formed strand is continuous.

lagging strand will bing with the primers, DNA poly adds Okazaki fragments, leading to a discontinuous replication because of disjointment

continuous and discontinuous strand have been formed, an exonuclease removes all RNA primers and replaced with the bases, another exonuclease proofreads

DNA ligase joins Okazaki fragments together forming a single unified strand.

ends of each strand has telomeres, repeated sequences of DNA that act as protective caps

telomerase, an enzyme, will catalyze the synthesis of telomere sequences at the end of the DNA

unwinds and rewinds DNA strand to prevent the DNA from becoming tangled or supercoiled

a single base substitution

a small DNA segment is lost

a segment of DNA is added

modification of the reading frame after a deletion or insertion, resulting in all codons downstream being different

the process where gene info is used to make a function gene product that will make protein as the end product

a gene is translated as it is transcibed

no nucleus, so newly made mRNA is directly accessible to ribosomes

no introns, no splicing of mRNA

a nuclear membrane separates the process of transcription and translation

mRNA has to move to the cytoplasm after splicing

steps:

1. RNA pol transcribes RNA from DNA

2. Introns are excised, exons are spliced together to from mRNA

3. mRNA out of nucleus to cytoplasm where ribosomal subunits bind to it

4. tRNA molecules attach to amino acids and go to ribosome

5. tRNA + amino acids → A site

6. peptide bonds form in P site and tRNAs exit at the E site

7. grows until polypeptide is complete

the process of creating a complementary RNA copy of a sequence of DNA

steps:

1. initiation: RNA pol binds to promoter of a gene and begins to unwind

2. elongation: RNA pol reads 3’ to 5’ and adds complementary ribonucleotides

   1. termination: RNA pol hits stop signal or falls off

mRNA from transcription is decoded by the ribosome to make an amino acid chain or polypeptide that will be a proteinc

one amino acid has multiple codons

each codon specified the amino acid to placed at the corresponding position along a polypeptide

carries info on AA sequences of proteins from DNA to ribosomes

serves as a translator molecule in protein synthesis; mRNA codons → amino acids

catalytic roles and structural roles in ribosome

precursor to mRNA, rRNA, tRNA before being processed, can catalyze its own splicing

structural catalytic role in spliceosomes, protein and RNA that splice pre-mRNA

A,P,E sites important for gene expression, two subunits

1. mRNA leaves nucleus and goes to ribsomes

2. mRNA brings small subunit

3. tRNA bring amino acid to ribosome and anticodon tRNA binds to codon mRNA

4. amino acids bind and form polypeptide

5. free tRNA is released

6. other tRNAs bring amino acids to the ribosome

GFP with a phenolate anion in the chromophore (EX: EGFP [Enhanced Green Fluorescent Protein])

The most common mutation to cause ionization of the phenol of the chromophore is when Ser65 is replaced by Thr (mutation S65T)

These mutations are set to improve the folding efficiency, not to increase the brightness (even though this could be an indirect effect).

GFP with a neutral phenol in the chromophore

When T203I largely suppresses the 475 nm peak, there is a singular peak at 399 nm, meaning that the chromophore will be neutral in almost all the ground-state molecules.

which is beneficial because you can have a more specific signal as well as reduced overlap to clearly see which light is emitted/absorbed.

GFP’s chromophore’s phenolate ion has an aromatic ring stacked next to it (T203F).

YFP

GFP protein where Tyr66 is replaced with Trp (Y66W), making the chromophore have an indole instead of a phenol.

CFP (cyan)

One characteristic of these (unexplained) is that there have double-humped excitation and emission peaks

GFP protein having a His instead of a Tyr6 (Y66H), making the chromophore have an imidazole instead of a phenol

This class has a low fluorescence quantum yield and is easily susceptible to photobleaching. There is also a EBFP (Enhanced BFP) that has emission and excitation peaks close to the regular BFP, but slightly lower. Enhanced BFPs will be more photostable and have a brighter intensity.

GFP protein having Phe at 66 instead of Tyr (Tyr66 for the wild type), the mutation being Y66F

shortest wavelengths obtained for a GFP.

If there are more gene copies and more promoters, there will be more protein present in the cell. It was studied that mammalian codons present will not hurt the expression levels present in other organisms, as seen with the additional valine or alanine in from of the initial methionine. In turn, GFP can be expressed freely, under the right conditions (highly specific), the protein folding autonomously. For example, after denaturing the protein, it could refold on its own into a cylindrical structure (chromophore must be in the middle, surrounded by the shell to properly function as a GFP).

Folding is a process that takes place under the right conditions, and the general mechanism is that GFP folds, following by the imidazolinone forming via nucleophilic attack on the Gly67 on the residue 65, followed by dehydration, which requires oxygen. Fluorescence cannot occur without atmospheric oxygen and by dehydrating the a-b bond in residue 66, the imidazolinone is in conjunction with the aromatic group. The presence of chaperones can help the protein fold, as well as mutations present in the GFP (especially Enhanced GFPS). The more efficient the fold, the faster and more reliable the reaction will be.

Once the protein is mature, oxygen is no longer required. The oxidation appears to be the rate-limiting step in the formation of this protein. However, it has not been studied on how to utilize the oxidative properties in the GFP, but it can be hypothesized that with more oxygen added, there will not be a faster reaction (with more oxygen present, we don’t breathe faster, so why would the GFP be any different? Not sure how right this is, just taking a guess).

Since the conditions to make GFP are super specific, the conditions to keep it active and functioning are similar. Under environmental duress, the protein may denature and unfold. Under the right conditions, if they return, the protein can refold, but must be rehydrated in order to be fully functioning.

tags and indicators, used as a tracker for what you are looking for

ex: C. elegans expression pattern for mechanosensory neurons

FRET, calcium sensitivity, dimerization

Fluorescence Resonance Energy Transfer (FRET) is a technique that takes two fluorophores with an overlapping emission (donor) and excitation (acceptor) spectrum. Highly dependent on distance, need to be close distance (<100 A).

o   One method of FRET is protease action where a BFP (class 6) is fused to GFP (class 2). The donor BFP has an emission spectrum peaking at 447 nm and it overlaps with the excitation spectrum of the GFP, peaking at 489 nm.

o   Disadvantage: high background signal that struggles to detect trace interactions in early forming cellular components.