Overview of Transcription

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Alexandrina Cornelia Rice
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Overview of Transcription The general process is

1 Overview of Transcription The general process is similar in prokaryotes and eukaryotes. Three phases: - Initiation The word gene was coined in 1909 (W. Johannsen). The central dogma (1950s). - Elongation - Termination In prokaryotes a singe RNA polymerase transcribes genes encoding mrna, rrna and trna.

Transcription and translation is coupled in prokaryotes 1) mrna are processed in

eukaryotes and prokaryotes Initiation of transcription Sense strand nontemplate

to it and begin translation on the 5 end of the molecule following right behind

2 Transcription and translation is coupled in prokaryotes 1) mrna are processed in eukaryotes but not in prokaryotes 2) rrna and trna are processed both in eukaryotes and prokaryotes Initiation of transcription Sense strand nontemplate strand = mrna 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. Antisense strand template strand 1 2 3

Pribnow (1975) P.N.A.S. -10 sequence double-stranded DNA separation.

3 Conserved sequences in prokaryotic promoters Pribnow box (TATA box) 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). D. Pribnow (1975) P.N.A.S. -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.

The moderately matched lacz promoter has about 1% initiation efficiency compared with ideal, and effective expression is strongly dependent

4 Promoter quality has a strong influence on the level of expression. Some examples of σ 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). Conserved sequences in eukaryotic promoters T T G A C A T A T A A T Promoter consensus sequences

5 The effects of mutations in the promoters region on transcription for the mouse β-globin gene. not recovered mutations (the relative transcription level was not determined) * more than one mutation was recovered for the corresponding nucleotide 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.

6 DNaseI Footprinting DNase I footprint performed on end-labelled DNA fragment FIS

7 Primer extension mrna 5 3 annealing + primer- 32 P G A T C Early-log Mid-log Late-log 37 C 10 C 37 C 10 C 37 C 10 C mrna 5 primer - 32 P 3 reverse transcriptase mrna 5 3 primer - 32 P cdna run on denaturating gel +77 cspa mrna

8 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).

Many of them are engaged in transcription; probably 2000 5000 enzymes are synthesizing RNA at any one time.

9 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 nucleotides added. About 7000 RNA polymerase molecules are present in an E. coli cell. Many of them are engaged in transcription; probably enzymes are synthesizing RNA at any one time. The typical eubacterial RNA polymerase consists of an essential four-subunit core enzyme organized as ααββ'. A fifth subunit ω interacts with and stabilizes β'. Deletion of the rpoz gene expressing ω results in a slow- growth phenotype. The α subunit (36.5 kda, rpoa gene) is organized in two domains, with the N-terminal (1-235) in contact with β or β' subunits. A flexible linker connects to the C-terminal domain ( , α-ctd) which lies outside the core polymerase and is the target for interaction both exit with activating factors such as Catabolite Activator Protein (CAP) and entry site site cis up-elements. The β subunit (150 kda, rpob gene) contains nine highly conserved regions and crosslinks to the 5'-end of nascent RNA. The β' subunit (155 kda, rpoc gene) contains eight conserved regions, contains the catalytic site, cross linking to the 3'-end of nascent RNA. The rudder is a projecting loop of the β' subunit which is proposed to act to separate the nascent RNA from the DNA template.

RNA polymerase passes through several steps prior elongation. 1) The enzyme remains at the promoter while it synthesizes the first ~10 nucleotide bonds.

10 RNA polymerase passes through several steps prior elongation. 1) 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 (< 10 nucleotides), releases them, and then starts synthesis of RNA again. 15 bp 2) 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 the flap segment of subunit β 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 reaction cycles. The hybrid DNA-RNA is only 8-9 nt long.

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

11 Initiation requires tight binding only to particular sequences (promoters), while elongation requires close association with all sequences that the enzyme encounters during transcription. 1) The core enzyme has a general affinity for DNA, (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 is reduced by a factor of ~10 4, and the half-life of the complex is <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. The binding constants extend from ~10 12 to ~10 6 M -1, reflecting promoter strengths that support initiation frequencies of ~1/sec (rrna genes) to ~1/30 min (the laci promoter).

that the bound sequence is directly displaced by another sequence.

12 Transition in shape and size identifies three forms of complex The RNA polymerase directly recognizes the promoter 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 moves along DNA

RNA polymerase approaches DNA from one side and recognizes that face of the DNA Prior modification experiments identify all those sites that the enzyme must recognize in order to bind.

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

Sigma factor (MW = 72000) Fragments 2.1 and 2.2, which map to a small helical region in σ 70, bind strongly to β'. Adjacent helical segments located in fragments 2.3 and 2.

2) of σ 70 were found to be inhibitory to DNA binding. The addition of σ to the polymerase core gives the RNA polymerase holoenzyme recognizing a site at -10 to form the closed complex.

14 Sigma factor (MW = 72000) Fragments 2.1 and 2.2, which map to a small helical region in σ 70, bind strongly to β'. Adjacent helical segments located in fragments 2.3 and 2.4 are involved in recognition of the -10 region of the promoter and binding the single stranded region of the transcription bubble. In addition, sequences near the N-terminal (1.1 and 1.2) of σ 70 were found to be inhibitory to DNA binding. The addition of σ 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 at the C- terminal end of σ, 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 σ at region 2.3, which binds to the non-template strand. Region 2.4 interacts with the upstream fork, and region 2.5 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.

Cambiamenti strutturali che avvengono nella RNA polimerasi durante l isomerizzazione (transizione complesso chiuso-complesso-aperto) -11

1 del fattore sigma sigma. Quando l oloenzima non è legato ad un promotore la regione σ 1.

1 è spostata 50 A fuori dall enzima permettendo l ingresso del DNA nel sito attivo. La regione σ 1.

15 Cambiamenti strutturali che avvengono nella RNA polimerasi durante l isomerizzazione (transizione complesso chiuso-complesso-aperto) -11 (template) (non template) +3 - Le pinze (pincers) bloccano il DNA nel complesso chiuso. - Cambiamento di posizione della regione 1.1 del fattore sigma sigma. Quando l oloenzima non è legato ad un promotore la regione σ 1.1 impegna il sito attivo bloccando l accesso al DNA. Quando si forma il complesso aperto la regione σ 1.1 è spostata 50 A fuori dall enzima permettendo l ingresso del DNA nel sito attivo. La regione σ 1.1 mina il DNA in quanto è carica negativamente. Il sito attivo sull enzima che interagisce alternativamente con il DNA o con σ 1.1, è carico positivamente. Alternative sigma factors respond to general environmental changes. Intrinsic DNA curvature TGNCCATA(C/A)T -10

16 The production of new sigma factors occurs during infection of B. subtilis by bacteriophage SPO1. α 2 ββ σ 43 gp28 gp33 gp34

Elongation 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.

17 Elongation 1) The antisense strand of DNA is used as template. 2) Transcription proceeds in 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. TFIIS avoids that RNA polymerase II stalls at certain nucleotide sequences.

Termination in prokaryotes 1) Core enzyme can terminate in vitro at certain sites in the absence of any other factor. These sites are called intrinsic terminators.

18 Termination in prokaryotes 1) 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 loop stem 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.

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

19 2) Rho dependent termination. -rho factor is an essential protein in E. coli (~275 kd) hexamer of identical subunits). -Mutations in rpob gene (β subunit of RNA polymerase) can reduce termination at a rho-dependent site. A consensus sequence for rho-dependent terminators cannot be defined (high C and low G content). - E. coli has relatively few rhodependent 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 about the relationship between transcription and translation. The RNA polymerase pauses when it reaches a terminator, and termination occurs if rho catches it there. Stop codon

genes in the unit. This effect is called polarity.

20 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. UAA UGA UAG

Transcription is the first step of gene expression, in which a particular segment of DNA is copied into RNA by the enzyme, RNA polymerase.

Transcription in Bacteria Transcription in Bacteria Transcription in Bacteria Transcription is the first step of gene expression, in which a particular segment of DNA is copied into RNA by the enzyme,