Gene Expression

Prokaryotic Gene Transcription

G & G Chapter 29

9/14/11Thomas Ryan, Ph.D.

Biochemistry and Molecular Geneticstryan@uab.edu

Modified Central Dogma of Francis Crick (1958)

Prokaryotic Chromosome (E. coli)

• Large circular chromosome - 4.6 x 106 bp

• Genome forms a compact structure called the nucleoid

• DNA organized in 50-100 loops (domains)

• The ends of loops are constrained by binding to protein structure which is in contact with cell membrane

All cells transcribe 3 major types of RNA molecules:messenger RNA (mRNA)ribosomal RNA (rRNA)transfer RNA (tRNA)

In archea and eubacteria:3 RNAs are produced by a single DNA dependent RNA polymerase

In eukaryotes:3 RNAs are produced by 3 distinct DNA dependent RNA polymerases (I, II, & III)

Additional Forms of RNA

rRNA & tRNA (stable): Not degraded rapidly (although extensively processed) Rapidly growly E. coli:

80% of RNA is rRNA 15% is tRNA

Ribosome number [rRNA] is proportional to growth rate

mRNA (translated): ~2 to 5% of total RNA is mRNA unstable with a t1/2 ≈ 2-3 min - allows regulation at the

level of mRNA synthesis

RNA Quantity and Stability

mRNA Degradation by RNases

Exonucleases (3’—> 5’ only in bacteria)

5’ 3’

Endonucleases (internal cuts)

5’ 3’

Transcription: The Players

Ribonucleotides (NTPs)

Template (DNA)

DNA Dependent RNA Polymerase

Transcription factors

Transcription: DNA to mRNAG & G page 907

Major bases found in DNA and RNA

DNA RNA

Adenine Adenine Cytosine Cytosine Guanine Guanine Thymine Uracil (U)

uracil-adenine base pairthymine-adenine base pair

DNA Dependent RNA Polymerase: Catalysis Reaction

Growing RNAstrand

Templatestrand DNA

n+1

A T A T

C GC G

n

Direction of synthesis is 5’ to

3’ No primer required Template required

Chromosome is divided into genes, which encode RNA and protein products.

Chemical differences ribose vs. deoxyribose*uracil vs. thymine

single stranded complementary to one strand of DNA (bottom in this case)

Transcription

AGUC5’ 3’RNA*

5’ AGTC 3’3’ TCAG 5’

DNA [ ]Gene

Naming the DNA Strands of a Gene

5’ AGTC 3’3’ TCAG 5’[ ]

Transcription

AGUC5’ 3’

DNA

RNA

topnontemplatenontranscribed

bottomtemplatetranscribed

5’ 3’3’ 5’[ ]

Transcription

5’ 3’

DNA

RNA # 1

[ ]Gene # 1

Gene # 2

P

PTranscription

3’ 5’RNA # 2

Both DNA Strands Encode Genes And Can Be Transcribed.

Prokaryotic RNA polymerase

• Synthesizes all major classes of RNA messenger RNA (mRNA) ribosomal RNA (rRNA) transfer RNA (tRNA)

• Multisubunit Protein• Holoenzyme = a2bb' catalyzes initiation of RNA

synthesis specifically at a promoter• Core enzyme= a2bb' catalyzes elongation of the

RNA chain

Transcription in Prokaryotes Only a single RNA polymerase

In E.coli, RNA polymerase is 465 kD complex, with 2 a, 1 b, 1 b', 1

a subunits appear to be essential for assembly and for activation of enzyme by regulatory proteins

b binds NTPs, interacts with , and forms catalytic site with b'

b' binds nonspecifically to DNA and forms catalytic site with b

recognizes promoter sequences on DNA, aids in melting the dsDNA by binding nontemplate strand

Prokaryotic Transcription Cycle Initiation

Holoenzyme binds to the promoter, unwinds DNA, and forms phosphodiester bonds between 7 to 12 nucleotides

Need to recognize promoter Elongation

dissociates Core enzyme elongates RNA with high processivity

Termination Polymerase dissociates from template DNA and releases

new RNA Rho()-factor dependent or independent.

Binding of RNA Polymerase to Template DNA

• Polymerase binds nonspecifically to DNA with low affinity and migrates, looking for promoter

• Sigma () subunit recognizes promoter sequence • RNA polymerase holoenzyme and promoter form

"closed promoter complex" (DNA not unwound) - Kd = 10-6 to 10-9 M

• Polymerase unwinds about 14 base pairs of DNA to form "open promoter complex" - Kd = 10-14 M

RNA Polymerase Binding to DNA - Promoter Search

Nonspecific binding to DNA: holo - Ka ≈ 107/M(i.e., to non-promoter DNA) (very rough numbers)

Specific binding to DNA: holo - Ka ≈ 1014/M(i.e., to promoter) (actual value depends on promoter!)

Note: in E. coli there are: ~3000 molecules of RNAP core~1000 molecules of ~1000 promotersvirtually unlimited nonspecific DNA sites

Holoenzyme searches for promoters by sliding along DNA and by intramolecular transfer on the chromosome.

Properties of Promoters See Figure 29.3

• 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 • The Pribnow box near -10, with consensus TATAAT

- this region is ideal for unwinding - why?

Prokaryotic PromotersG & G Fig. 29.3

Consensus Factor Promoters

Stages of Transcription

See G & G Figure 29.2 • binding of RNA polymerase holoenzyme

at promoter sites • initiation of polymerization • chain elongation • chain termination

Transcriptional Events

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 7-12 unit oligonucleotide has been made, sigma

subunit dissociates, completing "initiation" • Note mode of action of rifamycin (rifampicin)--binds to b

subunit of RNA polymerase and blocks first phosphodiester bond. Specific for prokaryotic RNA polymerase!

Events at initiation of

transcription

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

The Elongation Complex

• RNAP core enzyme covers about 60 bp of DNA, with about 17 bp unwound = transcription bubble.

• The bubble must contact the active site for polymerization.

• At the beginning of the bubble, the DNA is unwound, implicating a helicase activity.

• At the end of the bubble, the DNA is rewound.

Supercoiling Versus TranscriptionG & G Fig. 29.4

b subunit of RNAPIntercalates G:C basepairs

Inhibitors of Transcription

Transcription 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– “Intrinsic Termination”

Transcription TerminationSequence Dependent / Factor Independent

(Intrinsic Termination)

Stem Loop Structure

Transcription Termination: Rho-factor Dependent

Regulation occurs at every level

Transcription (RNA synthesis)RNA stability, processing, localization

TranslationPost-translational

Regulation of Prokaryotic Gene Transcription

Regulation of Prokaryotic Gene TranscriptionIntroduction

DNA:protein, protein:protein interactionsOrganization of genes into operons lac and trp operonsPositive control or activationNegative control or repressionAttenuation control of transcription

promoter = DNA site recognized by RNA polymerase for specific transcriptional initiation

terminator = region of DNA containing signals for termination of transcription

structural gene = DNA that encodes a protein (or RNA product?)

cistron = gene, mRNA specified by the structural gene

coding region = structural gene or cistron

open reading frame (ORF) = coding region (i.e., no stop codons)

operon = promoter + (gene)n + terminator, where n ≥11 transcript ≥ 1 cistron

Transcription Terminology

3D structure of regulatory proteins - most of them are homodimers

DNA sequence recognized by homodimers are typically palindromic (inverted repeats); they have dyad symmetry

Each monomer of the homodimer is in contact with bases in half of the palindromic sequence

This allows the protein coding region to remain relatively small while the protein recognizes a large sequence that is quite specific

General Rules for DNA Binding Proteins

Transcription factors

DNA binding proteins that decrease (repressors) or increase (activators) the efficiency of transcription at the promoter.

Transcription is the primary site of control in prokaryotes

Promoters drive the expression of genes

Prokaryotic genes can be arranged in operons

Transcription Regulation in Prokaryotes

• Genes encoding for enzymes of metabolic pathways are grouped in clusters on the chromosome - called operons

• This allows coordinated regulation and gene expression

• A regulatory sequence adjacent to such a unit determines whether it is transcribed - this is the ‘operator’

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

Operators can be upstream, downstream, or overlapping with the promoter.

General Organization of Operons

Regulatory proteins that bind to the operator can influence the access of RNA polymerase to the promoter thereby affecting the rate of transcription initiation.

Coordinate Regulation • Expression of several or numerous genes can be

controlled simultaneously.• Operon: a set of genes that are transcribed from the

same promoter and controlled by the same operator site and regulatory proteins.

• Regulon: a set of genes (and/or operons) expressed from separate promoter sites, but controlled by the same regulatory molecule. Global regulons may coordinate expression of many genes and operons, and may induce some, but repress others.

Global Regulation Via Sigma FactorsDifferent promoter architectures are recognized by

sigma factor subunit of RNA polymerase

Gene Expression

Prokaryotic Gene Transcription(Cont.)

&Eukaryotic Transcription

Histones and Chromatin

G & G Pages 336-340, Chapter 29,

9/14/11Thomas Ryan, Ph.D.

Biochemistry and Molecular Geneticstryan@uab.edu