L4 - Transcription, Translation, & Protein Signaling

is gene expression tightly regulated?

yes

what does the central dogma of molecular biology describe?

making proteins

what 3 polymerases involved in gene expression differ in template specificity, location w/i nucleus, and susceptibility to inhibitors?

RNA Polymerase I, II, & III

what dos RNA polymerase I make?

18S, 5.8S, & 28S ribosomal RNA

what does RNA polymerase II make?

makes pre-mRNA, & several small RNAs

what does RNA polymerase III make?

makes 5.8S rRNA & tRNAs

what is rRNA?

ribosomal RNA

what is tRNA?

transfer RNA

what is miRNA?

micro RNA

what kind of RNA is translated into proteins?

mRNA

what RNA can play roles in gene expression & protein synthesis?

all RNA (but only mRNA gets translated into proteins)

how long due most human mRNAs exist for?

1-10 hours before they are cleaved by cellular nucleases

how is RNA made?

made on a DNA template by RNA polymerases at transcriptional start sites

what is a consensus sequence?

short sequence of nucleotides which is found several times in the genome & is thought to play same role in its different locations (protein binding site)

what is the TATA box considered?

a non-coding DNA sequence (it contains consensus sequence characterized by repeating T & A base pairs)

what do promoters contain?

contain consensus sequence to bind transcription factor (TF) & polymerase

what are the gene regulatory elements?

promoters, enhancer, activators

what is an enhancer?

DNA sequence that promotes transcription, each one is made up of short DNA sequences called distal control elements

why do activators bind to distal control elements?

to interact w/ mediator proteins & TFs

what protein complex regulates the amount of mRNA produced?

RNA Polymerase II

does eukaryotic mRNA processing happen before or after the mRNA transcript is produced?

after, involves multiple steps

why is capping important?

regulates nuclear export (cap needed to transport through nuclear pore), prevents degradation by exonucleases (capped mRNA look like 3’ end of RNA), promotes translation (translation initiation factor 4E binds to capped mRNA)

what is the mRNA capping structure?

methylated guanine nucleotide that’s linked w/ triphosphates to the 5’ nucleotide instead of via phosphodiester bond

what are the general steps of processing eukaryotic mRNA?

capping 5’ end, adding poly-A tail to 3’ end, splicing, mRNA decay

what is splicing?

removal of introns & joining of exons

what is the normal phosphodiester bond like?

linkage between the 3’ carbon atom of 1 sugar & the 5’ carbon atom of another

what does the poly-A tail consist of?

multiple adenosine monophosphates (stretch of RNA that has only adenosine bases)

what does the poly-A tail do to the RNA molecule?

makes it more stable, prevents its degradation, allows mature messenger RNA to be exported from nucleus & translated into a protein by ribosomes in cytoplasm

what is alternative splicing?

form of transcriptional regulation, enables production of different mRNA isoforms from a single genes, mRNA is then translated to produce different proteins

what are the 4 enzymes involved in the mRNA decay step of processing eukaryotic mRNA?

deadenylase, decapping enzyme, 5’ endonuclase, 3’ endonuclease (all are required for completed degradation of mRNA)

what is the key process in the regulation of gene expression?

eliminate mRNA when there is enough protein

what is RNA degradation crucial for?

elimination of defective RNAs

what does translation require from tRNA?

to bring AAs to form a growing peptide chain

how are mRNA codons read?

from 5’ to 3’

how is a protein synthesized?

from N-terminus to C-terminus (extruded through a channel in the ribosome’s large subunit)

what is the start codon?

AUG (encodes for methionine)

what are the stop codons?

UGA, UAG, UAA (don’t encode for AAs)

how many ribonucleotides make up 1 codon?

3

what do codons dictate?

specific AAs or stop/start sequence

is formation of charged tRNA required for protein translation?

yes

what is the 1st step of mRNA translation to make a protein?

aminoacyl tRNA synthetase attaches AAs to their corresponding tRNA molecules using energy from ATP

what kind of bonds are individual AAs linked w/ to make a protein?

peptide bonds

what kind of free group does the N terminus have?

free amino group

what kind of free group does the C terminus have?

free carboxyl group

what is the difference between ribonucleotides & deoxyribonucleotides?

sugar component is ribose/deoxyribose (hydroxyl vs hydrogen at carbon 2)

what are some ribonucleotides?

adenosine, guanosine, uridine, cytodine

what are the purine molecules?

adenosine & guanosine

how is 5-phosphoribosyl-1-pyrophosphate used?

R5P receives PPi using PRPP synthetase. PRPP reacts w/ glutamine via amidotransferase to build purine ring (for pyrimidines, orotate phosphoribosyl transferase adds PRPP to ring

is methionine produced from one of the 5 starting families?

no

which AA is produced from 3-phosphoglycerate?

makes serine

does the synthesis of alanine require aminotransferase?

yes

which alpha-keto acid receives an alpha amino group to generate alanine?

pyruvate receives an alpha-amino group, then this is paired w/ a second aminotransferase rxn

what sites w/i ribosomes accept tRNA?

A & P sites

what is the E site in ribosomes?

exit

what is the P site in ribosomes?

peptidyl site for tRNA binding

what is the A site in ribosomes?

aminoacyl/acceptor site for tRNA binding

what are the parts of the subunits of ribosomes that accept tRNA?

large ribosomal unit consists of E/P/A sites, small ribosomal sububit consists of mRNA binding site

what do the ribosomes help tRNA do?

bring in appropriate AAs for peptide synthesis

how are proteins marked for degradation?

by adding 1 or more ubiquitin proteins to the degradation target

what is the process of marking a protein for degradation called?

ubiquitination or ubiquitylation

what molecules are proteins degraded by?

26S proteasome

what are secondary ubiquitin molecules always linked to?

1 of the 7 lysine residues or the N terminal methionine of the previous ubiquitin molecule

is ubiquitin recycled/reused in protein degradation?

yes

what is ubiquitin?

small protein that exists in all eukaryotic cells

what does ubiquitin consist of?

76 AAs & has molecular mass of about 8.6 kDA

what is the function of ubiquitin?

to perform its myriad functions via conjugation to a large range of target proteins, a large variety of modifications can occur (degradation is an example)

what does the 26S proteasome do?

protein complexes which degrade unneeded/damaged proteins by proteolysis

what is proteolysis?

chemical rxn that breaks peptide bonds

what kind of enzymes help proteolysis rxns?

proteases

what are the 6 types of receptors that mediate in intracellular signaling?

G protein coupled receptor, receptor tyrosine kinase, receptor guanylyl cyclase, gated ion channel, nuclear receptor, adhesion receptor (integrin)

explain G protein coupled receptor during signal transduction

external ligand binds to receptor & activates an intracellular GTP binding protein, which regulates an enzyme that generates an intracellular second messenger

explain receptor tyrosine kinase during signal transduction

ligand binding activates tyrosine kinase activity via autophosphorylation

explain receptor guanylyl cyclase during signal transduction

ligand binding to extracellular domain stimulates formation of 2nd messenger cyclic GMP

explain adhesion receptor (integrin) during signal transduction

binds molecules in extracellular matrix, changes conformation, which alters its interactions w/ cytoskeleton

explain gated ion channel during signal transduction

opens/closes in response to concentration of signal ligand or membrane potential

explain nuclear receptor during signal transduction

steroid binding allows the receptor to regulate the expression of specific genes

describe GPCR in detail

several transmembrane helices, extracellular binding site for ligand, intracellular binding site for GTPase (G protein trimer, aby subunits)

what does GTP binding do to GPCRs?

induces a conformational change dissociating Gα (active) from Gβγ, GTP to GDP hydrolysis turns off G protein & causes reassociation, when on the G protein activates adenylyl/guanylyl cyclases

describe receptor tyrosine kinases in detail

single transmembrane segment catalytic receptors, 1 transmembrane domain, has internal domain w/ a tyrosine kinase activity (catalyzes phosphorylation of select tyrosine residues in target proteins using ATP), signal is relayed to effectors of membrane transport/gene transcription/protein synthesis

are G protein coupled receptor & G protein linked receptor the same?

yes

are G protein & G protein coupled receptor the same?

no, 2 different proteins

what are transducers?

second messengers, pass on the signal

what second messengers does G protein activity produce?

cAMP, phospholipid derivatives, release of Ca2+ ions

what is activated when phosphorylation cascades by receptor tyrosine kinases are activated?

other protein kinases, protein phosphatases

what are some consequences of conformational changes and signal transduction?

open ion channels, form nuclear transcription complexes

explain Ca2+ & calmodulin as second messengers

Ca2+ is second messenger & calmodulin is its modulating protein, that is a transducer in that it regulates

what are the steps of Ca2+ & calmodulin as second messengers?

calcium channel open, calcium enters & binds to calmodulin & causes conformational change, change is required for binding to another protein

does the cytosolic Ca2+ ion concentration increase or decrease when the calcium channel opens?

increases (in the opening of calcium channels in membranes of calciosomes, endoplasmic reticulum, & plasma membrane)

how many calcium modulated proteins are known?

about 180

how can cytoplasmic calcium ions be increased?

stimulated by cAMP, stimulated by inositol-1,4,5-P3 (IP3)

are kinases catalytic receptors?

yes