bi203 exam 3 beffert

boston university 2025-2026

chapter 8 translation + translational regulation

whats the fundamental machinery of translation, how do we regulate

transcription basis

getting mRNA ready for translation

codons

nucleotides read in triplets

is aug the first set of nucleotides in mRNA

no - always a 5’ utr at start and 3’ utr at end

codon reading direction

5’ to 3’ - dnap and rnap synthesize same direction, read backwards

mRNA stranded

single

how many recombinations of aminoacids

61

carboxy terminus at which end?

3'

part btwn start and stop codons

open reading frame

nucleotide sequence to aminoacid sequence

TRANSLATE between ‘different languages’ - helped by tRNAs

tRNA anticodon

antiparallel to the mRNA - running 3’ to 5’

tRNA details

70-90 nucleotides, not identical/derived from different genes (400 genes to about 40-50 tRNAs), form cloverleaf structure

tRNA nucleotides modification reasons

folding and stability - folding makes it stable so it doesnt degrade after one use

3’ overhang

cca site, aminoacid attaches to this site. rna + protein makes tRNA with aa a different kind of molecule/hybrid

RNApol iii

transcribes tRNA

pre-tRNA transcript

acgu, undergoes processing to generate mature trna

how does trna receive specific aminoacid

enzymes whose job it is to recognize both the trna and the aminoacid - histodine is transfered by hystidyl trna synthetase

charged trna

combination aminoacid and trna, ready for translation. can be recycled cause it gives up aminoacid, the trna can be used again

aminoacyl trna synthetases

name for big family of aminoacid transfering enzymes - around 20 different enzymes, each one recognizes at least one trna/more than one trna

nonstandard pairings btwn mrna - trna

g-u, because of how ribosome lines up mrna - trna. ribosome sets up the molecule so that compl. base pairs adhere to wobble effect, and third nucleotide is g-u (weaker/more loose). others are inosine paired w u, c, a

wobble effect

first 2 nucleotides are super strong so the thirds strength doesnt matter as much when determining the aminoacid

inosine

modified adenosine, only in trna

ribosome

80s. enzyme that makes the peptide bond btwn carboxy group and amino group of 2 aminoacids. dehydration rxn, covalent bond. synthesis amino → carboxy. attached proteins changing all the time

large subunit

60s - made of 28S, 5s and 5.8s rRNA. forms peptide bond. made of rRNA and proteins.

small subunit

40s - made of 18S rRNA. recognizes where the start is, uses atp in the scanning process

RNApol i ribosome

nucleolus, covers 28s, 18s, 5s

rnapol iii ribosome

nucleus, covers 5.8s

pre rRNA transcript

self splicing, processing, 5s by rnapol3 → combined into different ribosomal subunits

translation in prokaryotes

rnapol transcribes into mrna, ribosomes just jump on to any aug and translate. no capping/tails, no nucleus, no exporting mrna etc. “all of these molecules live in the same soup”

lac operon

single mrna for multiple genes - polycistronic

poly-cistronic

most bacterial mrnas, so efficient that theres more than one proein from one mrna

mono-cistronic

one gene, one protein

how does ribosome know which aug in bacteria?

aggaggu (shine-dalgarno sequence) upstream of aug, tells ribosome to start translation here. recognized by small bacterial subunit that rRNA has a complimentary sequence to. only then recruits large subunit

translation stages

initiation - finds aug, recruits large subunit

elongation - pull mrna through ribosome and read nucleotides in sets of 3

termination - when stop codon reached, polypeptide is released and ribosome dissociates/ribosomal subunits are recycled

how to initiate translation

EIFs (eukaryotic initiation factors)

eif2

binds methianyl trna - first aa of every protein

poly-a binding protein

poly-a tail regulates translation rates. if you dont have a tail, poly-a binding protein cant interact with the tail and cant form initiation complex - CANT TRANSLATE

eif4E

binds 5’ methyl cap, similar to polya binding protein. checks does it have a cap, if not, doesnt translate

unwind the mrna

bumpy road, some eifs smooth it out. requires energy - atp dependent process

scanning of aug

small subunit looks for aud, finds it and hydrolyzed eif2, uses energy from eif2 to stuff the trna into large subunit and start translation. ‘glue’ is eif5b, combines large and small subunit

gtp vs gdp

gtp is active, gdp inactive. triphosphate vs diphosphate. if gdp is bound to a protein its p much inactive

internal ribosome entry site (ires)

viruses jump in further than the 5’ methyl cap closer to the aug start site. some euk do this aswell. poliovirus. happens more when cells are stressed - lower energy, takes shortcuts. initiates translation independently of 5’ methy cap, saves energy due to less scanning

eukaryotic elongation factors (eef)

eif but elongation. incredible

first steps of peptide building

first methionine trna is snapped in by eif2, second aminoacid is put in by eef1alpha, moved one over/translocated by eef2 - loses phosphate when snapped in, gdp, inactive

e, p, a sites

large subunit. exit, peptidyl, aminoacyl sites

release factor

no trna for stop codon, protein the size of trna recognizes stop codon sequences. unbinds polypeptide, large subunit, small subunit, etc. literally everything 

small gtp binding proteins

gtp being bound is allosteric modulation - with gtp bound the eef1alpha is in a specific conformation, with gdp bound its in another. so gtp bound is active, gdp bound is inactive.

usually have gtpase activity - hydrolyzes gtp to use its energy

to be recycled, kicks off gdp and binds new gtp - NOT A PHOSPHORYLATION, replaces it via guanine exchange factors (GEF)

posttranslational modification

translational regulation of SPECIFIC mrna

global regulation of translation ALL mrna - cell has low energy

autophagy

recycling pathway

ferritin regulation irt iron levels

protein made when iron is present so that it can be stored - translational regulation. iron response element in the 5’ utr (~6 nucleotides), with enough iron it binds small ribosomal subunit and translation begins. not enough iron, irp (iron repressor protein) binds to block initiation factors. similarly in 3’ utr

regulation of specific mrna

ferritin vs lac operon

translational regulation (rna) vs transcriptional regulation (dna)

poly-a tail translation rates

proteins that shorten the poly-a tail bind to 3’ utr and shorten/extend the tail, poly-a binding protein binds and initiaties translation - transcription takes time, if we want quicker response, we regulate at translational level

regulation of specific mrna

RNAi

synthetic siRNAs can be used to target specific mRNA to block gene expression/regulate endogenous translation

regulation of specific mrna

miRNA repress translation via RNAi

perfect pairing - all 21 of miRNA nucleotides matches up with mRNA perfectly, cuts via RISC, knocks down mRNA perfectly. efficient

mismatched pairing - pairing isn’t perfect, doesnt cut. just hangs on to the mRNA. works as physical barrier, repression/deadenylation (poly-a tail gets shortened), THEN mRNA gets degraded. slower, less efficient.

regulation of specific mrna

regulation of initiation factors eIF2 and eIF4E

eIF2 brings tRNA to small subunit - if GEF (eIF2B) doesnt bring another GTP to replace the GDP, it doesnt have enough energy to stick the tRNA into the ribosome

eIF4E recognizes 5’ methyl cap - regulated by 4EBP (4e binding protein), blocks translation if present/not phosphorylated. to stop translation, we dont phosphorylate the repressor and it represses

chaperone proteins

stabilize unfolded polypeptide chains to prevent aggregation during transport to organelles - puts lipophilic proteins inside of the cell so that they dont stick to one another (lipids) and cytosolic chaperone brings protein to mitochondria and mitochondrial chaperone takes it from there

heatshock proteins - overexpressed at temperatures where proteins misfold cause theyre trying to fix the folding

why separate chaperones for cytosol and mitochondria?

pH!!!! mitochondrial matrix is around 8, outside is abt 7.5

chaperonin

subclass of chaperones, big barrels for proteins - provide isolated env for proteins to fold properly. energy required to put protein in/pull protein out

protein misfolding diseases

AD, parkinson’s, type 2 diabetes - amyloids, made up of beta sheet structures + tau proteins (cte - football players)

chaperones that help with protein folding

protein disulfide isomerase (pdi), endoplasmic reticulum, adds and breaks disulfide bonds (tries most stable conformation)

peptodyl prodlyl isomerase (ppi), proline cant twist so ppi helps it flip (cis/trans)

preproinsulin

pre-protein insulin, only used in exposure to sugar - activated protease cuts out the polypeptide keeping insulin inactive in the presence of sugar so that insulin active. posttranslational change

glycosylation

carb chains to proteins to form glycoproteins - sugar coating protects the protein from being degraded

glycosylated proteins

asparagine - n-linked sugars, endoplasmic reticulum

serine - o-linked sugars, golgi apparatus

lipid addition to proteins

cytosolic proteins → membrane-associated proteins, essentially lipid tails

n-myristolation - glycine, inner face

prenylation, palmitolation - cystine, inner face

GPI anchors - sticks phosphatidylinositol to outer face,

phosphorylated aminoacids

adding phosphate to aminoacids - serine, threonine, tyrosine

usually grouped as serine/threonine and tyrosine

recognition/consensus motifs

aminoacids surround hydroxyl group and make it stick out (recognition motif) so that the enzymes can phosphorylate them

motifs recognized arent the exact same, just similar shapes. the most recognized/favored is the consensus motif

erk docking sites

substrates elk-1 and atp (binds to active site of ERK)

phosphorylation conformational changes

exposes hidden active sites (phosphate is negatively charged, moves depending on the positive charge/dipole)

protein kinase A

usually two regulatory two catalytic subunits, cAMP binds and PKA activates, lets go of catalytic subunits bc of conformational change in regulatory subunits

proteasome

large multisubunit proteins (~60), barrel shaped core. 'lid complexes check for ubiquitin-tagged proteins, core has proteolytic enzymes that degrade those proteins. maintains protein homeostasis + cell cycle control, stress response, signal transduction (removal of ‘wrong’ proteins)

ubiquitin-proteosome pathway enzyme families

three families of enzymes - e1, e2, e3 ubiquitin ligase

moves ubiquitin tag from one protein to the other until sent for degredation

e1 activates ubiquitin, e2 is the ubiquitin-conjugating enzyme, works with e3 - e3 links e2 to the target protein and transfers the ubiquitin

2 e1, 40 e2, 600 e3 - e3 is very specific

ubiquitin-proteosome pathway polyubiquitin chains

mark proteins for degradation and get recognized by proteasome complex/“lid”, atp hydrolysis unfolds tagged protein and puts it into the proteasome core, deubiquitinase removes and recycles ubiquitin before protein gets degraded

cyclin b ubiquitination

when cyclin b is getting degraded, hits a low enough level that cdk1 inactivates, this causes cell to exit mitosis and re-enter interphase

ubiquitination to regulate cell function

nucleus

dna replication, transcription (dna/rnapol), storage and information center, rna processing and ribonucleoprotein (rnp) assembly

nuclear envelope structure

inner + outer membranes

nuclear lamina

nuclear pore complex

inner and outer membranes

two phospholipid bilayers (inner and outer), space in between/perinuclear space is called the lumen

lumen of nuclear envelope is continuous with the lumen of the ER (endoplasmic reticulum)

why are ribosomes attached to the er?

rough er - the er processes the proteins that the ribosomes make [RETURN]

nuclear lamina

cytoskeletal structure holding it up, only structural protein inside nucleus SHAPE OF NUCLEUS - made of lamins (polypeptides), fibrous (can stick to itself and make bigger structures). can make a dimer/coiled coil structure → polymer (head to tail dimers) → higher order structure (side-to-side polymers) 

hgps

hutchinson gilford progeria syndrome - lamin gene mutation leads to premature aging

why do mutations in nuclear lamins lead to tissue specific disorders when theres nuclei in every cell?

mechanical stress - squish a cell - cell changes shape, so does nucleus. if mutation in protein the nucleus might not be able to adjust

abberant gene expression - certain parts of dna are associated with the lamina, if they cant exist there that may result in incomplete gene expression

mechanotransduction - movement of things through the lamin

protein-protein interactions - some proteins are only expressed in specific cell types, if those interact with lamin proteins, that can lead to cell specificity

how does lamina keep shape of nucleus

protein protein intrxns

addition of lipids

protein protein intrxns lamina

anchored to the inner membrane w protein protein interactions - LBR and emerin proteins also involved in premature aging (functionally common 

lbr and emerin are also intrxn with chromatin, keeps cerrtain dna molecules closer to nucleus - anchor

addition of lipids lamina

prenylation of emerin - adding a lipid anchor to cytosolic protein inserts it into the (inner) membrane

linc complex

SUN and KASH proteins connect lamina (in nucleus) with cytoskeleton (in cytosol) - keeps nucleus in certain shape and relative position

nuclear pore complex

huge channel that allows molecules in/out of nucleus. eightfold symmetry with big pore in middle, allows big molecules like ribosomal subunits to get in/out. made up of 30~ proteins called nucleoporins (NUPs), central channel is made up of FG nucleoporins (phenyl alanine-glycine)

passage of all molecules (going through double membrane is so tough and unnecessary when theres a big hole there)

what molecules go through nuclear pore complex

pH is the same, so small molecules (<20-40kD) can just go through - passive diffusion

larger proteins require energy dependent transport

how do proteins get identified for transfer

peptide signal on nuclear protein is nuclear localization signal (NLS) - T antigen protein (example) has a very specific sequence that when mutated didnt go to the nucleus (NECESSARY)

when that sequence was put on a different protein it was taken to the nucleus as well (SUFFICIENT)

pro lys lys lys arg lys val

can be bipartite (separated), rich in basic aminoacids

how do proteins get transfered to the nucleus

importin receptor binds to the NLS, takes it to the nuclear pore complex and ‘shoots them through the hoops’

regulated by small GTP binding protein called Ran (active when GTP)

lifecycle of Ran

generally inactive gdp bound in cytosol - low ran/gtp in cytosol, high ran/gtp in nucleus

brought in/out of nucleus, high ran/gtp in nucleus is because of the ran/gef in nucleus

ran/gap is on fibrils outside of nuclear pore complex, ran/gtp bumps into ran/gap, gets hydrolyzed Right There

how does ran cycle affect transport

importin binds the nls, transports cargo through nuclear pore. ran/gtp gets involved IN the nucleus, when importin runs into ran/gtp, lets go of target protein. now loose target protein and ran/gtp + importin leave nuclear pore and get jumped by ran/gap and ran/GTP becomes ran/GDP, lets go of importin, cycle repeats

ran controls export as well

exportin (opposite importin) and nuclear export signal (nes)

nes is recognized and bound by exportins, ran/gtp binds as well and transports it out of nucleus, jumped by ran/gap and becomes ran/gdp etc etc

karyopherins

transport receptors (importin/exportin)

how does ran/gdp get back in the nucleus

specific transporter NTF2

mrna transport

NO KARYOPHERINS - independent of ran/gtp

protein complex including poly-a binding protein moves mRNA through nuclear pore (in → out), helicase releases some proteins

one way trip. hello laika

snurportin

imports snrproteins, not super important but remember it uses importin - coming INTO nucleus

nfkappab

usually trapped in cytosol by IkB, needs to be in nucleus. signaling phosphorylation and ubiquitination of IkB allows IkB degrade + NFkB to nucleus to activate transcription

IMPORTANT

pho4

serine adjacent to its NLS is phosphorylated which blocks binding, dephosphorylation lets importin bind, pho4 transported to nucleus where it activates target gene transcription

how do we know where elements are located within chromosomes in the nucleus

3C (chromosome conformation capture) - take cells, fix everything in place, digest with restriction enzymes, ligate those cut bits of DNA, sequence/identify interxting regions - not linear anymore, but chromosome 2 next to chromosome 10 etc

euchromatin/heterochromatin

euchromatin is lighter, nucleus

heterochromatin is darker/denser, nuclear envelope and nucleolus

point of nucleolus

ribosomal rna synthesis, no membrane but bordered by heterochromatin