Cell Bio Review

A glucose molecule is to starch as

a nucleotide is to a nucleic acid

a shortage of phosphorus in the soil would make it especially difficult for a plant to manufacture

DNA

Lipids differ from other macromolecules in that they

are not true polymers

unsaturated fats

have double bonds in their fatty acid chains

which of the following is an organic molecule

a. o2

b. h20

c. hcl

d. ch4

CH4

monomers are attached together to create polymers when a hydroxyl group and a hydrogen atom are ___ in a _____

added; dehydration synthesis

which of these is not a lipid?

a. steroid

b. fat

c. polysaccharide

d. wax

polysaccharide

animals store their excess carbohydrates in the form of

glycogen

nearly all _____ are __________

enzymes; proteins

the difference between one amino acid and another is found in the

R group

which of the following is not a type of RNA?

a. nRNA

b. mRNA

c. rRNA

d. tRNA

nRNA

each amino acid in a polypeptide is specified by

a codon

which of the following statements is correct about the genetic code?

a. each codon encodes an amino acid

b. each amino acid is encoded by only one codon

c. a codon consists of three nucleotides

d. a codon and its complementary anticodon have the same sequences

C.

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

the process of obtaining a copy of the information in a gene as a strong of messenger RNA is called

transcription

the site where RNA polymerase attaches to the DNA molecules to start the formation of an RNA molecules is called

promoter

the process of taking the information on a strand of messenger RNA and building an amino acid chain, which will become all or part of a protein molecule, is called

translation

if an mRNA codon reads UAC, its complementary anticodon will be

ATG

Which of the following accurately describes gene expression in prokaryotic cells?

a. all genes are on all the time in all cells, making the needed amino acid sequences

b. some genes are always off unless a promoter turns them on

c. some genes are always on unless a promoter turns them off

d. some genes remain off as long as a repressor is bound

D

which of the following statements is correct about eukaryotic gene expression?

a. mRNAs must have introns spliced out

b. mRNAs contain the transcript of only one gene

c. enhancers act from a distance.

d. some genes remain off as long as a repressor is bound

C

which of the following is not a mechanism of controlling gene expression in eukaryotic cells

a. blocking translation with small interfering RNA

b. activating an enhancer

c. translating a gene as it is being transcribed

d. alternative splicing of the primary RNA transcript

B

dehydration synthesis

molecules form by removing water

hydrolysis

molecules bread down by adding water

primary structure

sequence of its amino acids

secondary structure

formed by hydrogen bonds between nonadjacent carboxyl and amino groups

tertiary structure

results from interactions between R-groups

quaternary structure

results from hydrogen and ionic bonds between separate tertiary structures

X ray crystallography

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

five different nitrogenous bases

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

difference between DNA and RNA structure

OH or H in sugar base

U or T

single strange or double strand

difference between DNA and RNA function

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

DNA stores the genetic information

A and T can align to form _____

two hydrogen bond

G and C can align to form ______

three hydrogen bonds

carbohydrate

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

lipids

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

fat → triglyceride

saturated fats

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

tightly packed, solid at room temp

unsaturated fatty acids

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

loosely packed, liquid at room temp]

central dogma

replication → DNA → transcription → RNA → translation → protein

DNA replication

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

1. replication fork formation

2. primer binding

3. elongation

4. termination

replication fork formation

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.

primer binding

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

elongation

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

termination (DNA rep)

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

topoisomerase or DNA gyrase

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

point mutations

a single base substitution

deletion

a small DNA segment is lost

insertion

a segment of DNA is added

frame-shift mutation

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

gene expession

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

gene expression in prokaryotes

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

gene expression in eukaryotes

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

transcription

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

translation

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

degeneracy

one amino acid has multiple codons

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

mRNA

carries info on AA sequences of proteins from DNA to ribosomes

tRNA

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

rRNA

catalytic roles and structural roles in ribosome

primary transcript

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

snRNA (small nuclear)

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

ribsome

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

steps in translation

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

cl

class 2 GFP

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).

class 3 GFP

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.

class 4 GFP

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

YFP

class 5 GFP

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

class 6 GFP

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.

class 7 GFP

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

shortest wavelengths obtained for a GFP.

expression

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).

formation

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.

maturation

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).

renaturation

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.

passive GFP applications

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

ex: C. elegans expression pattern for mechanosensory neurons

active GFP applications

FRET, calcium sensitivity, dimerization

FRET

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.