BSCI330 Exam 2

central dogma

information flow from DNA → RNA → Protein

amount of information content

DNA > mRNA > protein

does the central dogma apply to all cases of biological information?

no, there are well known exceptions (ex. telomerase and reverse transcriptase), but vast majority of cases follow central dogma

RNA transcription

generates a single-stranded RNA molecule that is complementary to the DNA template strand

RNA polymerase

a complex multi-subunit enzyme that synthesized RNA in a 5’ → 3’ direction (DNA read as 3’ → 5’)

number of RNA polymerases

eukaryotes have 3, prokaryotes have 1

promoters

special DNA sequences where transcription initiates

promoters in prokaryotes

RNA polymerase enzyme binds strongly to the promoter sequence

general transcription factors

help position RNA polymerase and start the process, required for transcription to start in eukaryotes

modification of chromatin structure

only in eukaryotes (prokaryotes don’t have chromatin), activator protein binds to an enhancer, allows for binding of general transcription factors, RNA polymerase, mediator, chromatin remodeling complexes, and histone acetylases, which then allows for full transcription to occur

terminator

sequence in prokaryotes where RNA transcription stops

polyadenylation signal

DNA sequence which stops transcription in eukaryotes

post-transcriptional processing

most eukaryotic RNA sequences require this before it can be functional, not found in prokaryotes

mRNA processing

required for RNAs that will encode proteins

first modification of mRNA processing

occurs immediately after 5’ end of RNA exits polymerase, addition of 7-methylguanosine “cap” to 5’ end of RNA, which marks RNA as an mRNA-to-be

5’ cap unique features

7-methylguanosine, 5’-5’ linkage, triphosphate bridge bond

introns

intervening sequences in most protein-coding genes that interrupt actual coding sequences

exons

expressed sequences/coding sequences

RNA splicing

process of removing introns

spliceosome

carries out RNA splicing, made up of small nuclear ribonucleoproteins (snRNPs) - small nuclear RNAs (snRNAs) and multiple proteins

process of RNA splicing

spliceosome assembles of pre-mRNA while it is still being transcribed, but splicing process may be delayed; given transcript may have many possible splicing patterns (ex. alpha-tropomyosin gene)

poly-A tail

attached on RNA 3’ end once transcription is complete, series of \~200 A’s that are added by a poly-A polymerase

poly-A binding proteins

bind to the poly-A tail, important for export from the nucleus and later protein synthesis

cytosol

location of protein synthesis (RNA synthesis and processing occurs in nucleus)

mRNA export process

once fully processed, mature mRNA is exported from the nucleus; removal of some proteins required (ex. snRNPs) and addition or retention of others (ex. poly-A binding proteins)

nuclear export receptor

binds mature mRNA and guides it through the nuclear pore complex into the cytosol

ribosomal RNA (rRNA)

up to 80% of cellular RNA, makes up structural and catalytic core of ribosomes

formation of rRNA

synthesis by RNA polymerase 1 and RNA polymerase 3 (not same as mRNA); heavily processed and assembled with ribosomal proteins in nucleolus

protein translation

turns mRNA into protein, done by the ribosome after mature mRNA has been exported to the cytosol

codon

each set of 3 nucleotides, with 61 of the 64 possible codons encoding for an amino acid (other 3 are stop codons UAA, UAG, and UGA)

what is the result of the “degenerate” nature of the genetic code?

for a given protein sequence there may be more than one RNA sequence

intermediary

allows for amino acids to interact with mRNA

transfer RNA (tRNA)

matched amino acids with codons, short RNAs with distinctive 3D structure; amino acid attaches to the 3’ end covalently

anticodon

complementary to the appropriate amino acid’s codon, found in tRNA in a loop formation

process of protein translation

amino acid is coupled to tRNA by aminoacyl-tRNA synthetase, with each amino acid having a distinct synthetase; amino acid activated by conjugation to AMP (energetically expensive, requires 2 ATPs); amino acid then transferred from AMP to tRNA; synthetase then proofreads for accuracy

protein synthesis direction

N-terminal → C-terminal (new amino acid added to C-terminal end of growing chain), same direction as transcription

peptide chain

attached to last tRNA that was added

aminoacyl tRNA

replaces an old tRNA and extends the chain by one residue

ribosome

decodes the RNA message, reads one codon at a time in mRNA from 5’ → 3’

ribozyme

catalytic function derived from RNA, not protein

methionine (Met)

codon AUG, where translation begins (sets the reading frame), uses a special initiator tRNA that is different from Met tRNA used for the rest of translation

elongation factors

Ef-Tu/Ef-G in prokaryotes, EF1/EF2 in eukaryotes, use GTPase activity to allow proofreading and to speed up ribosome translocation

how to peptide bonds form?

since the ribosome does not hydrolyze ATP, the energy to form a peptide bond comes from ATP hydrolyzed in the charging of tRNA (the attachment of the amino acid to the tRNA), which is physically and spatially distant from peptide bond formation

GTP hydrolysis

not often used to create chemical bonds, instead used to regulate the process (ATP hydrolysis often used to create chemical bonds)

stop codon

UAA, UAG, UGA; termination of translation when ribosome encounters

release factor

binds to the ribosome, causing the hydrolysis of the peptidyl tRNA, released the completed protein

does an in-frame stop codon always terminate translation?

no, there are circumstances where a stop codon is translated to insert an amino acid (ex. selenocysteine)

polyribosome (polysome)

several ribosomes translating simultaneously on a single mRNA, allows for higher efficiency and greater accuracy of translation

antibiotics

many are protein synthesis inhibitors that mainly affect prokaryotes, but sometimes also affect ribosomes in mitochondria and chloroplasts

primary protein structure

linear sequence of amino acid residues, determined by mRNA code, held together by peptide bonds

function of primary structure

in combination with an protein’s environment, determines secondary, tertiary, and quaternary structures

is the amino acid sequence of every protein identical to the genetically encoded primary sequence?

no, sequence can be changed after synthesis and folding via cleavage

secondary protein structure

folding and twisting of the peptide backbone, held together by weak H-bonds between carbonyl and amine groups in the backbone, R-groups stick out from backbone

alpha helix

secondary structure, rigid and cylindrical, forms when H-bonding occurs between a C=O and N-H groups that are 4 amino acids apart on the polypeptide backbone

beta sheet

secondary structure, flat/folded and sheet-like, forms when H-bonding occurs between a C=O and N-H groups on adjacent polypeptide chains, adjacent chains can be parallel (N-terminal → C-terminal) or antiparallel (opposite directions)

rigid proline residues

insert a kink in a protein’s backbone and disrupt secondary structures

tertiary protein structure

3D arrangement of secondary structures, mostly held together by noncovalent attractions between R-groups or between R-groups and the surrounding environment (ex. aqueous or hydrophobic lipid bilayer interior)

unstructured loops (random coils)

link secondary structures together

disulfide bonds

strong covalent bond that can form between cysteine residues to cross-link parts of the polypeptide backbone, help to stabilize the protein, do not generate tertiary structures

3D folding of proteins

results in structures that assume the lowest possible energy state

protein stability

depends on the free energy change between the folded and unfolded states, DeltaG = G(folded) - G(unfolded), proteins become more stable as G(unfolded) > G(folded)

chaperonins

provide an isolated chemical environment that allow for proteins to fold, helps proteins reach their lowest energy state

protein folding w/ chaperonins

protein binds to the chaperonin “cage” and enters it, a chaperonin “lid” seals the cage, the protein folds into its appropriate shape and is released

prions

unusual contagious neurological diseases where proteins adopt alternative folded states then influences its neighbor proteins to adapt this alternative state, proved by Stanley Prusiner in the 1980s

protein domain

region of the protein that folds essentially independently of other regions

functional region of protein

represented by a domain, proteins are modular

catalytic domain of diphtheria

domain A, inhibits host cell protein synthesis

hydrophobic domain of diphtheria

domain T, inserts into membranes

receptor binding domain of diphtheria

domain B, attaches to cell surface

motif

similar domains which occur in many related proteins

quaternary protein structure

arrangement of multiple tertiary protein structures, held together by weak bonds and some disulphide bonds (same bonds as tertiary structures)

homomers

identical subunit polypeptides

heteromers

different subunit polypeptides

post-translational modifications

allows for changing of protein structure and function

covalent modification

occurs on an amino acid side chain and changes its chemical properties

proteolytic cleavage

removes amino acids from the original translated sequence

phosphorylation

addition of a negatively charged phosphate group to the R-group of serine, threonine, tyrosine (bacteria can phosphorylate histidine)

phosphate in phosphorylation

comes from ATP, forms phosphorylated amino acid residue along with ADP

protein kinase

class of enzyme that catalyzes phosphorylation

protein phosphatases

class of enzyme that catalyzes the removal of phosphate (phosphorylation is reversible, but does not reform ATP, instead just forms a free phosphate)

effects of phosphorylation

phosphate group adds two negative charges to protein, which can drive major structural changes or may create a new recognition site that allows other proteins to bind to the phosphorylated protein

you are studying a protein that is turned on by phosphorylation at a specific serine residue, what do you expect would happen if the serine is mutated to aspartic acid?

depends, it could never turn on again, or always turn on, depending on whether or not the phosphate is necessary for the protein to turn on, or if simply negative charge is required

ubiquitylation

addition of ubiquitin, a small cytosolic protein that is covalently attached to another protein

purpose of ubiquitylation

serves as a tag that can either mark proteins for degradation or direct proteins to specific locations in the cell

ligands

molecules that bind to proteins, binding generally achieved by noncovalent bonds and is reversible

strength of ligand binding

since molecules are in constant motion and bumping into each other, protein binding must be strong enough to withstand the jolting of molecular motions

how to achieve binding strength

3D complementarity, formation of several noncovalent bonds (more bonds = greater strength)

3D ligand binding sites

amino acids that contribute to binding a ligand are often quite far apart on a protein’s primary sequence, but come together when the protein folds

K(on)

rate of forward (association) reactions that create the protein-ligand complex

K(off)

rate of backward (dissociation) reactions that breakdown the protein ligand complex

K(a)

measures the strength of binding of the protein-ligand complex, K(a) = K(on) / K(off)

K(d)

dissociation constant, K(d) = 1/K(a)

what is the effect of lower dissociation rates

lower K(d) value, stronger binding

guanine nucleotide exchange factors (GEFs)

positive regulators, catalyzes addition of phosphate to GDP to create GTP

GTPase activating proteins (GAPs)

accelerate hydrolysis of GTP, conversion of GTP back to GDP

cyclin dependent kinases

contains an activating phosphorylation event, an inhibitory phosphorylation event, and a cyclin that binds to another protein; all of these events occurring results in the protein being on

enzymes

catalytic proteins that speed up cellular reactions to allow life, and do not change the free energy from a reaction

limits of enzymes

cannot make a reaction occur spontaneously and cannot alter concentrations of reactants and products when at equilibrium, cannot extract more useful energy per mole of reactants (can only extract them faster)

effect of enzymes

determine nearly all the chemical transformations that make or break covalent bonds in cells, can lower activation energy for a catalyzed reaction

enzymes as a catalyst

only required in small amounts, must be left uncharged at the end of a reaction so it can cycle back to bind more substrate, catalyzes equally the forward and reverse reactions