Overview of Transcription

The word gene was coined in 1909 (W. Johannsen).

The central dogma (1950s).

The general process is similar in prokaryotes and eukaryotes.

Three phases:

- Initiation

- Elongation

- Termination

In prokaryotes a singe RNA polymerase transcribes genes encoding mRNA, rRNA and tRNA.

Transcription in E. coli and in Eucaryotes

Procaryotes Eucaryotes

Genes are grouped into operons Genes are not grouped in operons

mRNA may contain transcript of each mRNA contains only

several genes (poly-cistronic) transcript of a single gene

(mono-cistronic)

Transcription and translation are coupled. Transcription and translation are

Transcript is translated already during NOT coupled.

transcription. Transcription takes place

in nucleus, translation in cytosol.

Gene regulation takes place by Gene regulation via transcription

modification of transcription rate rate and by RNA-processing,

RNA stability etc.

mRNAs are not processed in prokaryotes mRNAs are processed in in eukaryotes (splicing , CAP, poly-A tail)

rRNA and tRNA are processed both in eukaryotes and prokaryotes

Transcription and translation is coupled in prokaryotes

As soon the growing mRNA chain separates from DNA, ribosomes attach to it and begin translation on the 5’ end of the molecule following right behind the RNA polymerase while it is transcribing the mRNA.

Initiation of transcription

Sense strand nontemplate strand = mRNA

Antisense strand template strand

1 2

3

Conserved sequences in prokaryotic promoters

Startpoint in most E. coli genes is an A (the distance of the startpoint from the TATA box may vary from 5 to 9 nucleotides).

-10 sequence double-stranded DNA separation.

-35 sequence initial binding of RNA polymerase.

spacer region (16-19 bp) it is important in maintaining the appropriate positions of the -10 and -35 elements.

Pribnow box

(TATA box)

D. Pribnow (1975) P.N.A.S.

Conserved sequences in eukaryotic promoters

Promoter quality has a strong influence on the level of expression. Some examples of 70 promoters are shown (red indicates positions which are conserved). The moderately matched lacZ promoter has about 1% initiation efficiency compared with ideal, and effective expression is strongly dependent on activation by CAP. The poorly matched lacI promoter is even less efficient (LacI repressor is present only at few copy per cell).

T T G A C A T A T A A T82 84 78 65 54 45 80 95 45 60 50 96 Promoter consensus sequences

Upstream of the CAAT box most eukaryotic promoters (genes encoding mRNA) have additional conserved sequences: CG box (GGGCGG) and CACCC box (GCCACACCC). Their role is still unclear.

not recovered mutations (the relative transcription level was not determined)

* more than one mutation was recovered for the corresponding nucleotide

The effects of mutations in the promoters region on transcription for the mouse -globin gene.

1. Prepare end-labeled DNA.

2. Bind protein.

3. Mild digestion with DNAse I (randomly cleaves ds DNA on each strand)

4. Separate DNA fragments on denaturing acrylamide gels.

DNAse I Footprinting

Footprint

Samples in lanes 2-4 had increasing amounts of the DNA-binding protein (lambda protein cII); lane 1 had no protein.

DNase I footprint performed on an end-labeled DNA

fragmentFIS

DNase I

DNA-protein complex

Products of DNase I digestion are primer extended by linear PCR using a 5’ end-labeled oligonucleotide

Sequencing gel

Partially DNase I digested DNA is subjected to linear PCR

Gel ShiftElectro Mobility Shift Assay (EMSA)Band Shift

Incubating a purified protein, or a complex mixture of

proteins e.g. nuclear or cell extract, with a 32P end-

labelled DNA fragment containing the putative

protein binding site (from promoter region).

Reaction products are then analysed on a non-

denaturing polyacrylamide gel.

The specificity of the DNA-binding protein for the

putative binding site is established by competition

experiments using DNA fragments or

oligonucleotides containing a binding site for the

protein of interest, or other unrelated DNA

sequences.

* *

Non-denaturing PAGE

Retarded mobility due to protein binding

Free DNA probe

No protein add protein

Gel retardation assay

EMSA

virB virF virG

Evaluating the Binding Affinity

Bound DNA

Free DNA

Primer extension

mRNA5’ 3’

mRNA5’ 3’

annealing

primer -32P

mRNA5’ 3’

primer -32P

reverse transcriptase

cDNA

run on denaturating gel

primer-32P

+1-10

G A T C

+24

+42

+77

Early-log Mid-log Late-log

37°C 10°C 37°C 10°C 37°C 10°C

cspA mRNA

S1 Mapping

La nucleasi S1 digerisce DNA o RNA a singolo filamento

[#]

[#]

[*]

[*]

Il sito d’inizio della trascrizione si trova a 300 basi dall’estremità 5’ marcata del frammento di DNA usato come sonda

Bacterial RNA polymerase

The overall reaction rate is ~40 nucleotides/second at 37°C (for the bacterial RNA polymerase); this is about the same as the rate of translation (15 amino acids/sec). One mistake occurs every 10000 nucleotides added.About 7000 RNA polymerase molecules are present in an E. coli cell. Many of them are engaged in transcription; probably 2000–5000 enzymes are synthesizing RNA at any one time.

The typical bacterial RNA polymerase consists of an essential four-subunit core enzyme organized as ’ (449 kd, about ½ size of DNA Pol III). A fifth subunit (rpoZ) interacts with and stabilizes ‘

The subunit (36.5 kDa, rpoA gene) is organized in two domains, with the N-terminal (1-235) in contact with or ' subunits. A flexible linker connects to the C-terminal domain (249-329, -CTD) which lies outside the core polymerase and is the target for interaction both with activating factors such as Catabolite Activator Protein (CAP) and cis up-elements.

The subunit (150 kDa, rpoB gene) and the ' subunit (155 kDa, rpoC gene) form the catalytic site.

The rudder is a projecting loop of the ' subunit which is proposed to act to separate the nascent RNA from the DNA template.

entry site

exit site

RNA polymerase passes through several steps prior elongation.

1) Core enzyme + Sigma factor (σ)→ Holoenzyme

2) The enzyme remains at the promoter while it synthesizes the first ~10 nucleotide bonds. The initiation phase is protracted by the occurrence of abortive events, in which the enzyme makes short transcripts (less than ~ 10 nucleotides), releases them, and then starts synthesis of RNA again.

3) The initiation phase ends when the enzyme succeeds in extending the chain and escapes from the promoter. Transition to the elongation complex involves partial dissociation of the holoenzyme. The sigma factor is left at the promoter complex, and the core RNA polymerase proceeds downstream. Conformational changes in the core enzyme result in β subunits clamping around the DNA, so that the polymerase never leaves the template. This is critical for processive transcription, since RNA polymerase can't resume synthesis of an incompletely transcribed gene, and must be assured of remaining bound for 104-105 reaction cycles.

15 bp

1) The core enzyme has a general affinity for DNA because of the electrostatic attraction between the basic protein and the nucleic acid. The complex core enzyme-DNA is stable, with a half-life for dissociation of the enzyme from DNA ~60 minutes. Core enzyme does not distinguish between promoters and other sequences of DNA.

2) The affinity of RNA polymerase for DNA in general is reduced by a factor of ~104, and the half-life of the complex is less than 1 second when sigma factor is bound to core enzyme.

3) In the presence of sigma factor, the holoenzyme binds to promoters very tightly, with an association constant increased from that of core enzyme by on average 1000 times and with a half-life of several hours.

4) There is wide variation in the rate at which the holoenzyme binds to different promoter sequences. Thebinding constants extend from ~1012 to ~106 M-1, reflecting promoter strengths that support initiation frequencies of ~1/sec (rRNA genes) to ~1/30 min (the lacI promoter).

Initiation requires tight binding only to particular sequences (promoters), while elongation requires close association with all sequences that the enzyme encounters during transcription.

When sigma When sigma unattached, unattached, hand is closedhand is closed

Transition in shape and size identifies three forms of complex

The most likely model is to suppose that the bound sequence is directly displaced by another sequence. The enzyme continues to exchange sequences until a promoter is found.

The RNA polymerase directly recognizes the promoter

The RNA polymerase moves along DNA

Prior modification experiments identify all those sites that the enzyme must recognize in order to bind.Protection experiments recognize all those sites that actually make contact in the binary complex

The regions at –35 and –10 contain most of the contact points for the enzyme. Within these regions, the same sets of positions tend both to prevent binding if previously modified, and to show increased or decreased susceptibility to modification after RNA polymerase binding.

RNA polymerase approaches DNA from one side and recognizes that face of the DNA

The sigma factor, σ70 (MW = 72000)Fragments 2.1 and 2.2 of 70 bind strongly to '. Adjacent helical segments located in fragments 2.3 and 2.4 are involved in recognition of the -10 region of the promoter. The 2.3 region is required for melting.

In addition, sequences near the N-terminal (1.1 and 1.2) of 70 were found to be inhibitory to DNA binding.

The addition of to the polymerase core gives the RNA polymerase holoenzyme recognizing a site at -10 to form the closed complex. In the holoenzyme form, an additional DNA binding domain of the region 4.2, become unmasked, and this recognizes a second site at -35, approximately 2 helical turns of DNA away. If the -35 site is recognized, the holoenzyme melts the region -11 to +3 in the DNA, giving the open complex, and the bubble is stabilized by the ssDNA binding domain of at region 2.3. The region 2.5 interacts with dsDNA from -11 to –17 (spacer region).

Melting of the transcription bubble admits the template strand to the catalytic site, allowing initiation to proceed.

- Le pinze (pincers) bloccano il DNA nel complesso aperto.- Cambiamento di posizione della regione 1.1 del fattore sigma sigma. Quando l’oloenzima non è legato ad un promotore la regione 1.1 impegna il sito attivo bloccando l’accesso al DNA. Quando si forma il complesso aperto la regione 1.1 è spostata 50 A° fuori dall’enzima permettendo l’ingresso del DNA nel sito attivo. La regione 1.1 mina il DNA in quanto è carica negativamente. Il sito attivo sull’enzima che interagisce alternativamente con il DNA o con 1.1, è carico positivamente.

Cambiamenti strutturali che avvengono nella RNA polimerasi durante l’ isomerizzazione (transizione complesso chiuso-complesso-aperto)

(template)

(non template)

-11

+3

• Sigma70 (rpoD) (-35)TTGACA (-10)TATAAT Primary sigma factor, or housekeeping sigma factor.

• Sigma54 (rpoN) (-35)CTGGCAC (-10)TTGCA Alternative sigma factor involved in transcribing nitrogen-regulated genes (among others).

• Sigma32 (rpoH) (-35)TNNCNCCCTTGAA (-10)CCCATNT Heat shock factor involved in activation of genes after heat shock.

• SigmaS (rpoS) intrinsic curvature (-10)TGNCCATA(C/A)T Alternative sigma factor transcribing genes of stationary phase of growth. Note the extended -10 element.

The use of different sigma factors gives E. coli flexibility in responding to different conditions.

Alternative sigma factors respond to general environmental changes

E. Coli sigma factors recognize promoters with different consensus sequences

The production of new sigma factors occurs during infection of B.

subtilis by bacteriophage SPO1

2’43

gp28

gp33gp34

gp28

gp34gp33

1) The antisense strand of DNA is used as template.

2) Transcription proceeds in 5’--3’ direction.

3) The double stranded RNA-DNA hybrid is very transient. At any given time during transcription, the number of nucleotides of RNA that remain paired with the DNA template may vary between 8 and 10.

Several proteins can affect the rate of elongation. NusA slows elongation when RNA polymerase encounters certain sequences keeping the rate of transcription similar to the rate of translation so that the ribosomes are able to follow on RNA molecule right behind the RNA polymerase.

Elongation

Termination in prokaryotes1) Core enzyme can terminate in vitro at certain sites in the absence of any other factor. These sites are called intrinsic terminators. 2) Rho-dependent terminators are defined by the need for addition of rho factor in vitro transcription assay.

1) Intrinsic terminator

The importance of the run of U bases is confirmed by making deletions that shorten this stretch; although the polymerase still pauses at the hairpin,it no longer terminates.

Stem (CG rich)

loop

2) Rho dependent termination.

-rho factor is an essential protein in E. coli (~275 kD) hexamer of identical subunits).

-Mutations in rpoB gene (subunit of RNA polymerase) can reduce termination at a rho-dependent site.

- E. coli has relatively few rho-dependent terminators; most of the known rho-dependent terminators are found in phage genomes.

- rho has a 5’-3’ helicase action that can cause an RNA-DNA hybrid to separate; hydrolysis of ATP is used to provide energy for the reaction.

- The idea that rho moves along RNA leads to an important prediction aboutthe relationship between transcription and translation. The RNA polymerase pauses when it reaches a terminator, and termination occurs if rho catches it there.

A consensus sequence for rho-dependent terminators cannot be defined (high C and low G content).

Stop codon

In some cases, a nonsense mutation in one gene of a transcription unit prevents the expression of subsequent genes in the unit. This effect is called polarity. Rho and NusA create a link between transcription and translation.

UAAUGAUAG