Chapter 21 (Part 2)

Transcriptional Regulation and

RNA Processing

Gene Expression

• Constitutive – Genes expressed in all cells (Housekeeping genes)

• Induced – Genes whose expression is regulated by environmental, developmental, or metabolic signals.

Regulation of Gene Expression

AAAAAA5’CAPmRNA

RNA Processing

RNA Degradation

Protein DegradationPost-translational modification

Activeenzyme

Transcriptional Regulation

• Regulation occurring at the initiation of transcription.

• Involves regulatory sequences present within the promoter region of a gene (cis-elements)

• Involves soluble protein factors (trans-acting factors) that promote (activators) or inhibit (repressors) binding of the RNA polymerase to the promoter

Cis-elements• Typically found in 5’

untranscribed region of the gene (promoter region).

• Can be specific sites for binding of activators or repressors.

• Position and orientation of cis element relative to transcriptional start site is usually fixed.

Enhancers• Enhancers are a class of cis-elements

that can be located either upstream or downstream of the promoter region (often a long distance away).

• Enhancers can also be present within the transcribed region of the gene.

• Enhancers can be inverted and still function

5’-ATGCATGC-3’ = 5’-CGTACGTA-3’

Activators and Repressors

RNAP

RNAP

Aco-A

• Activators and repressors- Bind to specific cis-elements

• Promote or inhibit assembly of transcriptional initiation complex.

• Co-activators and co-repressors – bind to proteins associated with cis-elements.

co-R

+1

+1

R

Structural Motifs in DNA-Binding Regulatory Proteins

• Crucial feature must be atomic contacts between protein residues and bases and sugar-phosphate backbone of DNA

• Most contacts are in the major groove of DNA • 80% of regulatory proteins can be assigned

to one of three classes: helix-turn-helix (HTH), zinc finger (Zn-finger) and leucine zipper (bZIP)

• In addition to DNA-binding domains, these proteins usually possess other domains that interact with other proteins

The Helix-Turn-Helix Motif

• contain two alpha helices separated by a loop with a beta turn

• The C-terminal helix fits in major groove of DNA; N-terminal helix stabilizes by hydrophobic interactions with C-terminal helix

The Zn-Finger Motif

Zn fingers form a folded beta strand and an alpha helix that fits into the DNA major groove.

The Leucine Zipper Motif

• Forms amphipathic alpha helix and a coiled-coil dimer

• Leucine zipper proteins dimerize, either as homo- or hetero-dimers

• The basic region is the DNA-recognition site

• Basic region is often modeled as a pair of helices that can wrap around the major groove

Transcription Regulation in Prokaryotes• Genes for enzymes for pathways are

grouped in clusters on the chromosome - called operons

• This allows coordinated expression• A regulatory sequence adjacent to such a

unit determines whether it is transcribed - this is the ‘operator’

• Regulatory proteins work with operators to control transcription of the genes

Induction and Repression

• Increased synthesis of genes in response to a metabolite is ‘induction’

• Decreased synthesis in response to a metabolite is ‘repression’

Binding of some trans-factors is regulated by allosteric

modification

lac operon

• Lac operon – encodes 3 proteins involved in galactosides uptake and catabolism.

• lacY = Permease – imports galactosides (lactose)

• lacZ = -galactosidase – Cleaves lactose to glucose and galactose.

• lacA = thiogalactoside transacetylase – function unclear

• Expression of lac operon is negatively regulated by the lacI protein

Lactose (Lac) Operon

• Diauxic growth• Operon organization• Negative and positive

regulation– Repressor (lacI)– CAP (crp)

Glucose is E. coli’s primary carbon source.

But.. it can grow on different carbon sources.

Diauxic growth of E. coli on a mixture of lactose + glucose.

• When Glucose runs out but Galactose is present, a set of genes (lac operon) are induced to break down Lactose

The lac I protein• The structural genes of the lac operon

are controlled by negative regulation• lacI gene product is the lac repressor• When the lacI protein binds to the lac

operator it prevents transcription• lac repressor – 2 domains - DNA binding

on N-term; C-term. binds inducer, forms tetramer.

Operator and RNA Polymerase Bind at Overlapping Sites

Inhibition of repression of lac operon by inducer

binding to lacI

• Binding of inducer to lacI cause allosteric change that prevents binding to the operator

• Inducer is allolactose which is formed when excess lactose is present.

Inducer : Allolactose,

produced by side reaction of lacZ Lehninger: Principles of Biochemistry, 3rd Ed.

IPTG is a Gratuitous Inducer

• Synthetic molecule

IPTG is a synthetic molecule Not metabolized by lacZUsed by molecular biologist to induce protein expression using the lac promoter

Binding of inducer causes allosteric

change that prevents binding

to operator

LacI is always bound to operator unless the inducer is present

Although some leaky expression does occur

Keeps lac operon in pressed state until it is needed

Repression of the

Tryptophan operon:

A variation of the theme

Catabolite Repression of lac Operon (Positive regulation)

• When excess glucose is present, the lac operon is repressed even in the presence of lactose.

• In the absence of glucose, the lac operon is induced.

• Absence of glucose results in the increase synthesis of cAMP

• cAMP binds to cAMP regulatory protein (CRP) (AKA CAP).

• When activated by cAMP, CRP binds to lac promoter and stimulates transcription.

Molecular Cell Biology, 4th Edition, Lodish et. al. (2000)

Why does the Lac Operon need an activator?

Lac promoter has lousy promoter!!!

Post-transcriptional Modification of RNA

• tRNA Processing• rRNA Processing• Eukaryotic mRNA Processing

tRNA Processing•tRNA is first transcribed by RNA •Polymerase III, is then processed•tRNAs are further processed in the chemical modification of bases

rRNA Processing•Multiple rRNAs are originally transcribed as single transcript.•In eukaryotes involves RNA polymerase I•5 endonuclases involved in the processing

Processing of Eukaryotic mRNA

5’ Capping• Primary transcripts (aka pre-mRNAs or

heterogeneous nuclear RNA) are usually first "capped" by a guanylyl group

• The reaction is catalyzed by guanylyl transferase

• Capping G residue is methylated at 7-position

• Additional methylations occur at 2'-O positions of next two residues and at 6-amino of the first adenine

• Modification required to increase mRNA stability

3'-Polyadenylylation

• Termination of transcription occurs only after RNA polymerase has transcribed past a consensus AAUAAA sequence - the poly(A)+ addition site

• 10-30 nucleotides past this site, a string of 100 to 200 adenine residues are added to the mRNA transcript - the poly(A)+ tail

• poly(A) polymerase adds these A residues

• poly(A) tail may govern stability of the mRNA

Splicing of Pre-mRNA • Pre-mRNA must be capped and polyadenylated

before splicing • In "splicing", the introns are excised and the

exons are sewn together to form mature mRNA • Splicing occurs only in the nucleus • The 5'-end of an intron in higher eukaryotes is

always GU and the 3'-end is always AG • All introns have a "branch site" 18 to 40

nucleotides upstream from 3'-splice site

Splicing of Pre-mRNA

• Lariat structure forms by interaction with 5’splice site G and 2’OH of A in the branch site.

• Exons are then joined and lariot is excised.

• Splicing complex includes snRNAs that are involved in identification of splice junctions.