MMBIO 240 - Exam Three

transcription

transcription

making RNA using DNA template

gene expression

process of using information in DNA to do something

RNA polymerase

enzyme that makes RNA from DNA template

transcription factor

molecule (protein) that regulates transcription

upstream and downstream

terms used to label gene sequences relative to base one

promotor

region of DNA directly upstream of start site; where transcription factors and RNA polymerase bind to initiate transcription

RNA synthesis

DNA structure determines RNA sequence through complementary pairing; U instead of T; ATP, CTP, GTP, and UTP required for RNA synthesis (2’ OH on sugar)

RNA chain grows in 5’ to 3’ direction; add onto 3’ OH like DNA synthesis

RNA polymerase doesn’t require any primer

Antisense strand serves as template for transcription

initiation

necessary proteins to appropriate locations on DNA, first nucleotides

+ one is the first DNA base to have RNA transcribed, negative is upstream and positive is downstream

core enzyme

enzyme sticks to DNA initially and polymerizes from some types of DNA, no discrimination on where to start transcription; five subunits α2ββ’ω

holoenzyme

core enzyme plus sigma factor; directs core enzyme to bind to specific gene promotors to transcribe certain genes: six subunits α2ββ’ωσ

elongation

polymerizing, adding onto chain 5’-3’ by moving 3’-5’ on template

termination

stop polymerization and detach proteins involved with DNA

Rho dependent termination

Rho protein binds at rut site, slides along transcript until it hits RNA polymerase and stops transcribing

Rho independent termination

Sequence that stops polymerization is part of newly produced RNA

hairpin

transcript filed to form termination hairpin; inverted repeat with a spacer; can act as a transcriptional terminator

inducible gene expression

gene normally inactive but can be activated, lac operon of E.coli as a model

repressible gene expression

gene normally active but can be deactivated, lac operon of E.coli as a model

constitutional gene expression

gene always transcribed regardless of conditions

open reading frame

DNA or RNA sequence that contains start or stop codon

monocistronic gene

DNA sequence that encodes a single ORF

polycistronic gene

single promotor that encodes multiple independent ORFs

operon

two or more contiguous ORFs under control of a single operator

operator

component of promotor that gives additional control over gene transcription

positive regulation

gene is inactive unless something induces gene expression

negative regulation

gene normally active unless repressor molecule binds to inactivate gene expression

betagalactosidase

converts lactose to galactose and glucose; easily measured lac operon controls lactose uptake and conversion to glucose can convert lactose to allolactose; permease and beta gal encoded in lac operon

permease

brings lactose into cell through membrane channel

transacetylase

transfers acetyl CoA to lactose, nonessential

repressor

keeps lac operon turned off

bacteria from nonlactose to lactose

cells adapt to use lactose as a food source to make glucose; make proteins needed to import lactose and break it down for energy production; rapidly detected by operons and polycistronic mRNAs because all proteins are in same mRNA

bacteria default conditions

lac operon turned off to conserve energy; repressor prevents unnecessary transcriptions, gene encoding repressor adjacent to genes encoding proteins to use lactose, but under separate promoter

allolactose

inducer binds to repressor, changes its shape, and blocks ability to bind to promotor or operator; only produced if lactose is present; turns on lac operon

leaky expression

receptor protein occasionally falls off operator, allowing a few molecules of permease and beta galactosidase present to initiate change; needed to make any allolactose

first ORF of polycistronic mRNA

translated before end of mRNA is finished; mRNA has short lifespan and may start degradation before transcription stops

low glucose levels

up regulates lac operon; not efficient to break down lactose if glucose is present, but lactose is broken down for energy when necessary

high cAMP

concentration increased by low glucose levels; signals starvation and changes gene expression; recruits RNA polymerase to promotors and increases transcription of genes; stimulates lac operon but can’t fully override repressor

trp operon

encodes necessary proteins and enzymes to synthesize tryptophan for use in making new proteins; turns off if tryptophan is already abundant

blocks at multiple levels: initiation is on or off; elongation and termination fine tune expression

trp operon level one

if tryptophan is present, it binds to repressor, allowing it to bind to promotor and operator and transcription is blocked; RNA polymerase cannot move past repressor protein to transcribe structural genes; reduces 70 fold

trp operon level two

intermediate levels of tryptophan synthesis; transcription has begun but the rate of ribosome movement on mRNA affects formation of secondary RNA structures and some terminate RNA polymerase; only in prokaryotes

RNA secondary structure

complementary RNA can potential base pair to make regions of double stranded DNA; anti parallel and complementary base pairing

attenuator

after transcription start site but before structural genes, at 3’ end of trpL sequence; functionality in RNA not DNA sequence; rho independent terminator so RNA polymerase falls off DNA copy when it runs into attenuator

a nucleotide sequence in DNA that can lead to premature termination of transcription

low concentration of tryptophan

ribosome moves slowly to wait for trp charged tRNAs, if covered, region one cannot pair, so a two to three pair loop occurs, forming anti terminator loop, transcription proceeds

high concentration of tryptophan

ribosome moves quickly across region one, allowing it to pair with region two, which allows a three to four loop to form transcriptional terminator; RNA polymerase falls off DNA to stop transcription, ribosome eventually falls off

RNA polymerase I

14 subunits, transcribe large rRNAs

RNA polymerase II

12 subunits, transcribe mRNA and noncoding RNA

RNA polymerase III

17 subunits, transcribe tRNA, small rRNA, other noncoding RNAs

bacteria RNA polymerase

core enzyme plus one of seven sigma factors, transcribes all RNAs

complexity of RNA polymerase

bacterial RNA polymerase are relatively simple, eukaryotic RNA polymerase has many accessory factors

binding of RNA polymerase

prokaryotic RNA polymerase directly binds to DNA promotor; eukaryotic RNA polymerase RNAP doesn’t bind to DNA directly and other factors recruit RNAP to specific DNA sites and transcription factors

nucleus

in prokaryotes, translation begins before transcription finishes and can regulate transcription by translational process; translation cannot occur in eukaryotes until mRNA leaves nucleus

chromatin

eukaryotes can use histone modifications to control transcription; prokaryotes have easy access to “naked” DNA for faster response and have fewer genes and fewer mechanisms needed to regulate gene expressions

mRNA processing in eukaryotes

five prime capping and three prime poly A tail addition for stability, export of mRNA to cytoplasm, translation promotion; splicing to remove unnecessary sequence and alternative splicing allows more coding potential

alternative splicing

exons can also be removed; single gene can be used differently to make many proteins

transcriptional regulation

specific DNA sequences usually upstream of transcription start site bind specific proteins which mediate gene expression; under different conditions different proteins produced to bind to same regions

study transcriptional regulation

clone regulatory regions and place just 5’ of a reporter genes to study promoters, operators, and enhancers; can artificially fuse one gene’s promotor to another gene’s coding sequence and produce protein; reporter genes easier to detect and quantify than protein encoded in gene

reporter assay

measures activity of various enhancer or promotor elements; luciferase takes a substrate and converts to visible light measured in a luminometer; more luciferase means promotor was more active

proteins in transcripts

clone cDNA into expression vectors by inserting into active non native promotor; opposite of studying transcription because you only want ORF not promotor

TATA box

TATAA most common sequence, about 25-30 bp upstream of start site, binds TATA binding protein, a component of TFIID to initiate transcription

initiator

InR, sequence surrounding and including start site of transcription, consensus: YYANA TYY; pyrimidine rich, Y C or T; A first base transcribed

downstream promotor element DPE: not present in every promotor, ~+28-+32

minimal pol II promotor

two elements, no TATA box but has DPE

primer extension

determines start site of RNA transcription; gene specific primer; assume sequence of entire gene is known but want to find first base transcribed

TATA binding protein

binds to TATA, TBP associated factors bind to initiator and downstream promotor element

TFIID

bind to promotor without assistance of other promotors, contains saddle shaped TBP which interacts with and causes conformational change in TBP and TATA in minor groove

TFIIA

stabilizes TFIID binding, prevents from falling off

TFIIB

binds complex and recruits RNA Pol II

TFIIF

comes with Pol II to help bind TFIIB

TFIIE

binds TFIIB and recruits TFIIH to complex

TFIIH

helicase and kinase activity to open template and phosphorylate RNA pol II

mediator complex

brings in RNAPII complex the first time and during subsequent rounds of transcription by interacting with activators

RNA Polymerase II C terminal domain

multiple copies of Tyr-Ser-Pro-Tyr-Ser Pro-Ser

CTD must be phosphorylated by TFIIH to trigger elongation

promotes capping, triggers start of elongation phase, plays a role in histone modification

transition from transcription initiation to elongation

nascent RNA transcript begins to push against TFIIB

RNA chain growth leads to TFIIB dissociation form polymerase

five prime end of ten nucleotide transcript no longer part of RNA DNA hybrid and begins to threat into exit tunnel

transcription complex escapes from promotor

TFIIB, TFIID, and TFIIA remain bound or near to promotor to facilitate subsequent rounds of transcription

transcription elongation complex

more stable than initiation complex, about fourteen bp melted to form transcription bubble, eight nucleotides within bubble paired with RNA chain, DNA opens in front of bubble and closes behind bubble as RNA polymerase moves along

RNA polymerase rate

unsteady, chain elongation temporarily delayed at pause sites, may lead to arrest and termination, arrest is a step in proofreading

basal transcription complex

inefficient at starting transcription; inefficient binding and rebinding to start over

structural motifs in DNA binding proteins

helix turn helix: two parallel helices with turn before third, third helix inserts in major groove

zinc fingers: peptide chain held together by zinc; homotrimer in major groove

leucine zippers

helix loop helix

activators

when bound to regulatory promotors, provides signals to move from transcriptional initiation to elogation; has independent domains

not bound to core promotor and gene specific

enhancer

promote transcriptions by strong recruiting of mediator complex; far from promotor, can be upstream or downstream and even in an intron, function when removed and reinserted in opposite direction, gene specific function

core promotors

near transcriptional start point; where RNA polymerase and transcription factors bind to initiate transcription; similar for all genes transcribed by RNA polymerase II due to same basal transcription complex

Regulatory Promotors

further upstream than core promotor, where transcriptional activator proteins bind and assist TFIID to recruit rest of basal transcriptional complex

basal factors

start transcription of all coding genes, TFIID, TFIIB, etc.

coactivators

do not bind to DNA directly but help distal DNA bound TF to interact with basal TFs at promotor

promotor/enhancer mechanism

activator proteins bind to regulatory promotors or enhancer when genes under their control are needed; mediator complex gets signal from activators to bring in RNAPII and basal factors to core promotor, activator bound to regulatory promotor makes contact with basal transcription complex and signals to transition from initiation phase to elongation

Silencers or negative regulatory elements

bind to repressors, interfere with basal complex formation or trigger increased condensation of DNA

insulator DNA

prevents enhancers from interacting with wrong promotor

DNA affinity chromatography

purification of DNA binding proteins

specific DNA sequences attach to beads in column; identifies DNA sequences that bind to any proteins AND specific proteins that bind to specific DNA

run SDS PAGE to determine what proteins, confirm with western blot

deletion of various promotor regions

identification of specific regions with regulatory activity by deleting DNA regions and analyzing enzymic activity of the various strands

linker scanning mutagenesis

replaces promoter sequences with other DNA sequences that have no regulatory function; preserves spacing of sequences

chIP assay

dependent on having antibody against target proteins

chromatin

DNA and associated proteins in a condensed state

Heterochromatin: more dense DNA, less accessible for RNA transcription, mostly repeat sequences, few genes, low activity, regular nucleosome array

euchromatin

less dense DNA, more available for RNA transcription, mostly gene sequences, potentially highly active genes, irregular nucleosomes

nucleosome modification

acetylation or methylation of basic amino acids in histone tails, removing positive charge makes DNA more loose

histone code

still being deciphered, critical area for gene expression manipulation and the prevention or treatment of diseases at the molecular level; acetylated histone tails likely to be active; phosphorylated tails prepare DNA for cell division

acetylases

add acetyl groups and activate genes, histone acetyltransferases

deacetylases

remove acetyl groups and deactivate genes, HDACs

chromatin remodeling

moves histones to new locations on DNA; displace from molecule or translocate to new position on same molecule, typically at initiator

partial dissociation of DNA from histone

DNA still associated, but RNAPII has easier path to transcribe DNA, modifications to weaken

Sliding Histone

expose DNA to enzymes involved in RNA transcription or DNA replication

detecting modifications with restriction enzymes

repositioning may expose new sequences to restriction enzymes; DNA purified after digested and inactivated from restriction enzyme

radioactive probe

monitor association of labeled DNA with histones; histones not normally free but the binding of radioactive DNA to histone indicates chromatin remodeling

epigenetics

patterns in gene expression that are controlled by heritable but potentially reversible changes in chromatin structure; as DNA replicates, new DNA still associated with same histone structure but don’t alter nucleotides