Transcription

and Regulation

of Gene

Expression

Outline

• Transcription in Prokaryotes

• Transcription in Eukaryotes

• Regulation of Transcription in Prokaryotes

• Transcription Regulation in Eukaryotes

• Structural Motifs in DNA-Binding proteins

• Post-Transcriptional Processing of mRNA

RNA

• Usually single-stranded

• Has uracil as a base

• Ribose as the sugar

• Carries protein-encoding information

•Can be catalytic

DNA

• Usually double-stranded

• Has thymine as a base

• Deoxyribose as the sugar

• Carries RNA-encoding information

• Not catalytic

Types of RNA

• mRNA, tRNA and rRNA

• These three kinds of RNA are present in

both eukaryotic and prokaryotic cells as

well as in mitochondria

The secondary structure RNA

• is a random coil, or is determined by its interaction

with proteins

• occasionally there are complementary regions

with the same RNA molecule

• LARGE DIFFERENCES IN PHYSICAL

PROPERTIES RNA AND DNA

• RNA - does not exist as a large double helix -

does not show hyperchromicity under denaturation

• does not have well-defined melting point

• show large changes in viscosity

Messenger RNAs (mRNA)

• mRNA carries the genetic information

coded in DNA into the cytoplasm

• The order of nucleotides in the mRNA

determines the order of amino acids in

the protein translated from it.

Transfer RNAs (tRNAs)

• This class of small RNAs

transfers amino acids to the protein-

synthesizing machinery and translates

the nucleic acid “language” into amino acid

“laguage”

Ribosomal RNAs (rRNAs)

• This class of RNAs plus a large number of

ribosomal proteins are assembled and make

up ribosomes, the enzymatic machinery

on which protein synthesis takes place.

• Ribosomes engage the mRNAs and form a

catalytic domain into which the tRNAs

enter with their attached AAs.

Other Forms of RNA

rRNA and tRNA only appreciated later

• All three forms participate in protein synthesis

• All made by DNA-dependent RNA polymerases

• This process is called transcription

• Not all genes encode proteins! Some encode

rRNAs or tRNAs

• Transcription is tightly regulated. Only 0.01% of

genes in a typical eukaryotic cell are undergoing

transcription at any given moment

TranscriptionThe new RNA molecule is formed by incorporating

nucleotides that are complementary to the template strand.

DNA coding strand

DNA template strand

DNA

5’

3’

5’

3’

G T C A T T C G G

C A G T A A G C C

G

RNA

5’

GG U C A U U C3’

RNA synthesis and processing

All 3 major classes of RNA rRNA, tRNA and mRNA:

are synthesized in the nucleolus (by copying of

DNA)

are modified after synthesis and before being

transported into the cytoplasm → PROTEIN

SYNTHESIS

the selection of the segment to be copied – is the

major control point in this process!

as synthesis proceeds, the RNA is released and

the DNA reforms a normal helical structure

Transcription in Prokaryotes Only a single RNA polymerase

• In E.coli, RNA polymerase is 465 kD complex,

with 2 , 1 , 1 ', 1

' binds DNA

binds NTPs and interacts with

recognizes promoter sequences on DNA

subunits appear to be essential for assembly

and for activation of enzyme by regulatory

proteins

• RNA polymerases contain no nuclease activity

Stages of Transcription

See next Figure

• binding of RNA polymerase holoenzyme

at promoter sites

• initiation of polymerization

• chain elongation

• chain termination

Properties of Promoters See Figure

• Promoters typically consist of 40 bp region

on the 5'-side of the transcription start site

• Two consensus sequence elements:

• The "-35 region", with consensus TTGACA

- sigma subunit appears to bind here

• The Pribnow box near -10, with consensus

TATAAT - this region is ideal for unwinding

- why?

Initiation of Polymerization • RNA polymerase has two binding sites for NTPs

• Initiation site prefers to binds ATP and GTP (most RNAs begin with a purine at 5'-end)

• Elongation site binds the second incoming NTP

• 3'-OH of first attacks alpha-P of second to form a new phosphoester bond (eliminating PPi)

• When 6-10 unit oligonucleotide has been made, sigma subunit dissociates, completing "initiation"

• Note rifamycin and rifampicin and their different modes of action

Chain Elongation Core polymerase - no sigma

• Polymerase is accurate - only about 1 error

in 10,000 bases

• Even this error rate is OK, since many

transcripts are made from each gene

• Elongation rate is 20-50 bases per second -

slower in G/C-rich regions (why??) and

faster elsewhere

• Topoisomerases precede and follow

polymerase to relieve supercoiling

Chain Termination Two mechanisms

• Rho - the termination factor protein

– rho is an ATP-dependent helicase

– it moves along RNA transcript, finds the "bubble", unwinds it and releases RNA chain

• Specific sequences - termination sites in DNA

– inverted repeat, rich in G:C, which forms a stem-loop in RNA transcript

– 6-8 As in DNA coding for Us in transcript

Transcription in Eukaryotes• RNA polymerases I, II and III transcribe rRNA,

mRNA and tRNA genes, respectively

• Pol III transcribes a few other RNAs as well

• All 3 are big, multimeric proteins (500-700 kD)

• All have 2 large subunits with sequences similar

to and ' in E.coli RNA polymerase, so

catalytic site may be conserved

• Pol II is most sensitive to -amanitin, an

octapeptide from Amanita phalloides

("destroying angel mushroom")

Transcription Factors More on this later, but a short note now

• The three polymerases (I, II and III) interact

with their promoters via so-called

transcription factors

• Transcription factors recognize and initiate

transcription at specific promoter sequences

• Some transcription factors (TFIIIA and TFIIIC

for RNA polymerase III) bind to specific

recognition sequences within the coding

region

RNA Polymerase II Most interesting because it regulates

synthesis of mRNA

• Yeast Pol II consists of 10 different peptides

(RPB1 - RPB10)

• RPB1 and RPB2 are homologous to E. coli RNA

polymerase and '

• RPB1 has DNA-binding site; RPB2 binds NTP

• RPB1 has C-terminal domain (CTD) or PTSPSYS

• 5 of these 7 have -OH, so this is a hydrophilic and

phosphorylatable site

More RNA Polymerase II

• CTD is essential and this domain may project away from the globular portion of the enzyme (up to 50 nm!)

• Only RNA Pol II whose CTD is NOT phosphorylated can initiate transcription

• TATA box (TATAAA) is a consensus promoter

• 7 general transcription factors are required

• See TFIID bound to TATA

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’

• Some substrates induce enzyme synthesis even though the enzymes can’t metabolize the substrate - these are ‘gratuitous inducers’ -such as IPTG

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

Alpha Helices and DNA

A perfect fit!

• A recurring feature of DNA-binding proteins

is the presence of -helical segments that fit

directly into the major groove of B-form DNA

• Diameter of helix is 1.2 nm

• Major groove of DNA is about 1.2 nm wide

and 0.6 to 0.8 nM deep

• Proteins can recognize specific sites in DNA

The Helix-Turn-Helix Motif First identified in 3 prokaryotic proteins

• two repressor proteins (Cro and cI) and the E.

coli catabolite activator protein (CAP)

• All these bind as dimers to dyad-symmetric

sites on DNA (see Figure)

• All 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

Helix-Turn-Helix II

See next Figures

• Residues 1-7 of the motif are the first helix

(but called "helix 2")

• Residue 9 is the turn maker - a Gly, of course

• Residues 12-20 are the second helix (called

"helix 3")

• Recognition of DNA sequence involves the

sides of base pairs that face the major groove

The Zn-Finger Motif

First discovered in TFIIIA from Xenopus laevis, the

African clawed toad

• Now known to exist in nearly all organisms

• Two main classes: C2H2 and Cx

• C2H2 domains consist of Cys-x2-Cys and His-x3-

His domains separated by at least 7-8 aas

• Cx domains consist of 4, 5 or 6 Cys residues

separated by various numbers of other residues

More Zn-Fingers Their secondary and tertiary structures

• C2H2 -type Zn fingers form a folded beta

strand and an alpha helix that fits into the

DNA major groove

• Cx-type Zn fingers consist of two mini-

domains of four Cys ligands to Zn followed

by an alpha helix: the first helix is DNA

• recognition helix, second helix packs

against the first

The Leucine Zipper Motif First found in C/EBP, a DNA-binding protein in

rat liver nuclei

• Now found in nearly all organisms

• Characteristic features: a 28-residue sequence

with Leu every 7th position and a "basic

region"

• (What do you know by now about 7-residue

repeats?)

• This suggests amphipathic alpha helix and a

coiled-coil dimer

The Structure of the Zipper

and its DNA complex

• Leucine zipper proteins (aka bZIP proteins)

dimerize, either as homo- or hetero-dimers

• The basic region is the DNA-recognition site

• Basic region is often modelled as a pair of

helices that can wrap around the major groove

• Homodimers recognize dyad-symmetric DNA

• Heterodimers recognize non-symmetric DNA

• Fos and Jun are classic bZIPs

Post-transcriptional Processing

of mRNA in Eukaryotes

• Translation closely follows transcription

in prokaryotes

• In eukaryotes, these processes are

separated - transcription in nucleus,

translation in cytoplasm

• On the way from nucleus to cytoplasm,

the mRNA is converted from "primary

transcript" to "mature mRNA"

Eukaryotic Genes are Split • Introns intervene between exons

• Examples: actin gene has 309-bp intron

separates first three amino acids and the other

350 or so

• But chicken pro-alpha-2 collagen gene is 40-

kbp long, with 51 exons of only 5 kbp total.

• The exons range in size from 45 to 249 bases

• Mechanism by which introns are excised and

exons are spliced together is complex and

must be precise

Capping and Methylation

• Primary transcripts (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

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

• Function not known for sure, but poly(A) tail

may govern stability of the mRNA

Splicing of Pre-mRNA Capped, polyadenylated RNA, in the form of a RNP

complex, is the substrate for 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

• Branch site is essential to splicing

The Branch site and Lariat • Branch site is usually YNYRAY, where Y =

pyrimidine, R = purine and N is anything

• The "lariat" a covalently closed loop of RNA is

formed by attachment of the 5'-P of the intron's

invariant 5'-G to the 2'-OH at the branch A site

• The exons then join, excising the lariat.

• The lariat is unstable; the 2'-5' phosphodiester is

quickly cleaved and intron is degraded in the

nucleus.

The Importance of snRNP • Small nuclear ribonucleoprotein particles -

snRNPs, pronounced "snurps" - are involved in

splicing

• A snRNP consists of a small RNA (100-200

bases long) and about 10 different proteins

• Some of the 10 proteins are general, some are

specific.

• snRNPs and pre-mRNA form the spliceosome

• Spliceosome is the size of ribosomes, and its

assembly requires ATP