Ribosomal RNA (rRNA)

Transfer RNA (tRNA)

Messenger RNA (mRNA)

single

3 distinct 

o   α 2, β, β’, Ϭ (=holoenzyme)

o   α 2, β, β’ (=core enzyme)

specificity factor

directs the core enzyme to transcribe specific genes

causes a tight binging between RNA polymerase and promoters

this stimulates the initiation of transcription

this can only occur in the presence of sigma

sigma can then do this...

melting of DNA and tight binding of RNAP

sigma can then dissociate from the RNAP and leave it tightly wrapped around the DNA and then move onto another RNAP

specific base sequences

RNA polymerase

cause it to bind

-10 box (pribnow)

6-7bp

10bp upstream

the start of transcription 

3 Fis sites between -60 and -150

no not bind to the RNAP

represent a group of transcription activating elements known as enhancers 

these help activate transcription without directly binding the RNAP

inhibitors of prokaryotic transcription initiation, but not elongation

do not inhibit initiation in eukaryotes 

occurs when the bacterial RNAP reaches a terminator at the end of the gene

there are two types:

Intrinsic terminators

Rho-dependent terminators

also called Rho-independent

presence of an inverted repated followed immediately by a T-rich region in the nontemplate strang

forms hairpin structure

model strong (efficient) terminator

associated poly(U) tail

trigger

RNAP to fall off from the template

rho is a helicase that unwinds RNA-DNA and RNA-RNA double helicases

requires the presence of a specific recognition sequences on the NEWLY SNYTHESIZED RNA

is a group of contiguous, coordinately controlled genes

lac operon is the classic example 

Galactoside permease (lac Y) - transport of lactose into cell

B-galactosidase (lac Z) - cleaves lactose to form galactose and glucose

Galactoside transcacetylase (lac A)

required to ultilize lactose

a single mRNA molecule from a single promoter

operon is turned on unless something is present to keep the control off

lac operon is off by the presence of lac repressor - its the regulatory gene

the repressor protein binds to the operator region (located downstream of promoter) and hinders the binding of RNAP

binding of the inducer causes a conformational change in the repressor, causing it to come off

now RNAP can bind to the promoter and lac operon can be transcribed 

the inducer molecule is a modified lactose

formed by a side reaction of B-galactosidase

repression is leaky

not total repression of the operon

very low, basal levels of lactose and lac operon products are always present 

lac operon is also under positive control

when there is too much glucose it turns off

achieved through catabolite repression

low glucose = raises cAMP concentration

lac operon transcription stimulated when CAP-cAMP complex binds to an activator

promotes binding of RNAP

lac is ________ since....

Trp is _______ since....

high levels of each does this...

catabolic, because it breaks down lactose

anabolic, because it synthesizes tryptophane

high levels stimulates lac, suppress trp

when there is too much tryptophan and the Trp operon needs to be suppressed

made of 2 gene loci - leader locus and attenuator locus

lead goes first and weakens transcroption causing a premature termination of transcription

after the attenuator region contains series of 8AT pairs in a row, inverted repeat, creates the hairpin

when tryptophan is short 
Charles Yanofsky proposed hypothesis

pretty much prokaryotes transcript/translate at the same time

Eukaryotic

RNAP I

most active at low ionic strength

stimulated by Mn and Mg

Nucleolar location

Eukaryotes

RNAP II

most active at high ionic strength

pronounced simulation by Mn

Nucleoplasmic location

Eukaryotic 

RNAP III

active over broad range of ionic strength

stimulated by Mn

nucleoplasmic location

RNAP I

synthesizes

hnRNAs

snRNAs

RNAP III

synthesize

5S rRNA precursor

tRNA precursors

U6 snRNA

7SL RNA

7SK RNA

recognized by RNAP I

associated with rRNA precursor

not well conserved

consists of a core element between positions -45 and +20

surrounds the transcription start site

and an upstream control element (UCE) between -156 and -107 

spacing between core and UCE determines strength of promoter 

recognized by RNAP II

two major components

Core Promoter and Upstream Element

Class II Promoters

Core Promoter

TATA Box

sequence of TATAAAA (non template)

~-30

housekeeping genes and developmentally regulated genes do not have

specialized genes (luxury genes) do

in general they help code start of transcription

Class II Promoters

Core Promoter

Initiator

conserved

located around the transcription start site required for optimum transcription

PyPy[start][any base] T

Class II Promoters

Core Promoter

Downstream Element

located downstream... duh

little is known...

Class II Promoters 

UpStream Element

also called GC Boxes and are GC rich

located upstream

stimulate transcription

there's also the CCAAT Box

both require require transcription factors to be stimulated

recognized by RNAPIII

transcribe variety of genes encoding for small RNAs

rRNA and tRNA are classical class III genes

are not directly part of the promoter but still strongly influence transcription

bind to DNA through interactions with other DNA binding proteins known as general transcription factos

transcription factors

enhancer-binding proteins

activators

TFIIA

TFIIB

TFIID

TFIIE

TFIIF

TFIIG

and TFII means transcription factor II

SL1

UBF (upstream binding factor)

TFIIIA

TFIIIB

TFIIIC

presence of histone proteins repress transcription (H2A, H2B, H3, H4)

acetylation of histone enhances transcription

accomplished through the action of HAT (histone acetyltransferase)

getting rid of the nonsence that RNAP synthesizes

pulls out introns sticks together exons

also splicers are the product of being addicted to Atom... which is a pretty awesome little deal

first two bp = GU

last two bp = AG

2'OH ground of A within intron reacts with phosphodiester bond between 1st exon and intron

exon 1 is released, lariat intron loops back to phosphiodiester of reacting A

exon 1 then butts out lariat by reacting at the same phosphodiester bond

the two exons are now buddies

particular places where splicing actually takes place

40S yeast and 60S human

composed of the lariat plus the particle

formation is ATP dependent and is required for efficient splicing

small nuclear ribonuclear proteins

recognize the consensus sequences at the ends and branch points of introns

assist in bringing together the ends of the conserved sequences to allow for splicing 

some don't need a spliceosome

these are ribosomes (surprise)

called catalytic RNAs

protection from degradation. resistance to RNAase due to triposphates linkages

confer enhanced translatability. allow the mRNA to associated with ribosome

facilitate export from the nucleus

increase efficiency of splicing

polyadenylation

poly(A)

the 3'ends of hnRNA and mRNA possess long chain of AMP residues

not made my a transcription of Ts but is added post-transcriptionally in the nucleus by poly(A) polymerase

confers stability to mRNA and stimulates translation

associates with polyadenylated mRNA and boosts translation

initiation

elongation

termination

tRNA charging to form aminoacyl-tRNA

dissociation of the prokaryotic 70S ribosome into its large (50S) and small (30S) subunits

tRNA charging

basic info

all tRNA is charged when it has covalently bound to an amino acid

all have the same base sequence at 3' end... CCA

an ester bond joins the carboxyl terminus of the amino acid to the 2'or3'-hydroxyl group of the terminal A of the tRNA

ultimately, the amino acid is attached to the 3' hydroxyl

Aminoacyl-tRNA synthatase catalyzes two reactions

reaction 1

amino acid is activated by coupling it with AMP to form aminoacyl-AMP

aa+AMP-->aminoacyl-AMP + pyrophosphate (PPi)

energy stored in the aminoacyl-AMP, thus activating it

Aminoacyl-tRNA synthetase catalyzes two reactions

second reaction

the stored energy is used to transfer the amino acid to the tRNA, forming aminoacyl-tRNA

aminoacyl-AMP + tRNA --> aminoacyl-tRNA + AMP

20

one for each amino acid

each synthetase exhibits a high degree of specificity 

dissociation of ribosomes

this step is necessary for inititation to occur as both pro and euk cells build translation initiation complexes on the small ribosomal subunits 

IF-1

IF-2

IF-3

IF1 and 3 participate in ribosome dissociation

IF-1 promotes dissociation of the 70S ribosome

IF-3 binds to the 30S subunit to prevent its reassociation with 50S

the dissociation of the intact ribosome is apart of ribosome cycling

each ribosome will dissociate into subunits at the end of each round of translation

all 3 IFs are bound; IF-1 can't do it by itself

the presence of the 3IFs allow for mRNA, aminoacyl-tRNA binding to occur

the first codon message is AUG - initiation codon 

formation of the 30S iniation complex

what AUG signals for

the binding of a special aminoacyl-tRNA that contains N-formyl-methionine (tRNA_f^met) or fMet-tRNA

fMet is the first amino acid incorporated into a polypeptide, but is typically removed during protein maturation

AUG

GUG
UUG
AUG

the mRNA contains a conserved sequence that is located upstream of start coon

called the Shine-Dalgarno sequence 

its bps are complimentary with what's located on the 3'end of the 30S ribosomal subunit of rRNA

GTP

1:1 GTP:fMet-tRNA

GTP is not hydrolyzed until 50S ribosomal subunit links with the complex to form the intact ribosome after the IF-2 has dissociated

requires that hte 50S and 30S subunits join

IF-1and2 dissociated from the complex

GTP hydrolyzes

IF-2 plus the 70S complex forms GTPase

initiation utilizes methionine rather than fMet

no Shine-Dalgarno sequence. the 5'cap directs inition factos to bind and search for the initiation codon

located at the 5'end of the message and scans downstream along the mRNA until the first AUG is found

has to be in the correct sequence: purine at -3, AG at +4, A (of AUG) at +1

Eukaryotic Initiation factors

eIF2

functions to bind the initiating aminoacyl-tRNA to the ribosome

requires GTP when eIF2 dissociated from the ribosome

Eukaryotic initiation factors

eIF2B

Eukaryotic initiation factors

eIF3

eukaryotic initiation factors

eIF4

eukaryotic initiation factors

eIF1 and eIF1A

eukaryotic initiation factors

eIF5

eukaryotic initiation factors

eIF6

in a 3 step process that is repeated over and over until the polypeptide being synthesized is complete

elongation

binding sites for aminoacyl-tRNAs

P (peptidyl) site

A (aminoacyl) site

fMet-tRNA is bound to the P site

elongation

step 1

fMet-tRNA (and AUG start) occupy the P site

second codon of the message occupies the A site and the matching aminoacyl-tRNA now also bind to the A site

the binding of aminoacyl-tRNA is dependent on elongation factor EF-Tu and GTP

part of the large subunit functions as a peptidyl transferase that removes fMet from tRNA in the P site and transfers it to the aminoacyl-tRNA occupying the A site

forms a peptide bond between fMet and amino acid in the A site (which is attached to that tRNA)

the P site now contains tRNA all alone

elongation

step 3

this is the translocation step

dependent on EF-G and GTP

the mRNA shifts position in the ribosome, causing the dipeptidyl-tRNA in teh A site to shift one codon length so it's not at the P site (it kicks out the other tRNA)

the process repeats 

goes until a stop codon is hit

really low thanks to proofreading process

incorrect codon/anticodon base paring results in a weak association between them

the incorrect tRNA has time to dissociate prior to GTP hydrolysis

termination

3 nonsence codons that are stop codons

UAG
UUA
UGA

tRNA doesn't recognise these

instead repease factor (RF) proteins do

termination 

repease factor 3 of them

RF-1 is specific for UAA and UAG

RF-2 is specific for UAA and UGA

RF-3 promotes binding of RF-1 and RF-2 to the ribosome and is the GTP-binding protein

induce the release of the completed polypeptide from the ribosome

tripeptide

Pro-Ala-Thr

pro binds to teh A uf UAG

Thr binds to the G of UAG

the tripeptide can also bind to UUA

tripeptide

Ser-Pro-Phe

Ser binds to the G of UGA

Phe binds to the A of UGA

RF-2 also binds to the UAA