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

1 Transcription in Bacteria

2 Transcription in Bacteria

3 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, RNA polymerase. If the gene transcribed encodes a protein, the result of transcription is messenger RNA (mrna), which then will be used to create that protein via the process of translation. Alternatively, the transcribed gene may encode for either noncoding RNA genes (such as microrna) or ribosomal RNA (rrna) or transfer RNA (trna), other components of the proteinassembly process, or other ribozymes.

4 Mechanism of transcription RNA polymerase is the enzyme that catalyzes RNA synthesis. Using DNA as a template, RNA polymerase joins, or polymerizes, nucleoside triphosphates (NTPs) by phosphodiester bonds from 5' to 3'. In bacteria there is one type of RNA polymerase and transcription and translation are coupled (they occur within a single cellular compartment). As soon as transcription of the mrna begins, ribosomes initiate protein synthesis. The whole process occurs within minutes.

5 Transcription in E. coli and in Eucaryotes Procaryotes Genes are grouped into operons Eucaryotes Genes are not grouped in operons mrna may contain transcript of several genes (poly-cistronic) each mrna contains only transcript of a single gene (mono-cistronic) Transcription and translation are coupled. Transcript is translated already during transcription. Transcription and translation are NOT coupled. Transcription takes place in nucleus, translation in cytosol. Gene regulation takes place by modification of transcription rate Gene regulation via transcription 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

6 Transcription and translation is coupled in prokaryotes Initiation of transcription Sense strand nontemplate strand = mrna Antisense strand template strand 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 3 1 2

7 How does transcription get started? RNA polymerase binds to a region on DNA called a PROMOTER Bacterial promoter structure Bacterial promoters are not absolutely conserved but they do have a consensus sequence. Conserved sequence: When sequences of DNA have exactly the same series of nucleotides in a given region. Consensus sequence Consensus sequence: there is some variation in the sequence but certain nucleotides are present at high frequency.

8 Bacterial promoter structure It is convention to indicate the start of transcription by the number +1 and to use positive numbers to count farther down the DNA in the direction of transcription (downstream). If transcription is proceeding to the right, then the direction to the left is called upstream with the bases indicated by negative numbers. For the majority of E. coli genes, the promoter consensus sequence consists of two hexamer sequences. Centered at 10 is the consensus sequence TATAAT, which is also known as the TATA box or Pribnow box. (capital letters indicate bases found in those positions in more than 50% of promoters analyzed; small letters = less than 50%).

9 Bacterial promoter structure Another region with similar sequences among many promoters is centered at 35. The consensus sequence at 35 is TTGACA. The spacing between the 10 and 35 sequences and the start point for transcription is important, and deletions or insertions that change the spacing are deleterious.

10 How does RNA polymerase recognize and bind to the promoter? Structure of bacterial RNA polymerase Comprised of a core enzyme plus a transcription factor called the sigma factor (σ). Together they form the complete, fully functional enzyme complex called the holoenzyme.

11 Structure of bacterial RNA polymerase The core enzyme The core enzyme is the component of the holoenzyme that catalyzes polymerization. It is 400 kd in size and has five subunits: two copies of the α-subunit (αi, αii), and one copy each of the β-, β -, and ω-subunits. X-ray crystallographic studies revealed a crab claw shape. Subunits α I, α II, and ω form the base of the claw, and subunits β and β form the pincers. These pincers form an internal channel (2.7 nm wide). The enzyme active site is located at this internal channel where an essential Mg 2+ ion is bound. The core enzyme has high affinity for most DNA. In the absence of σ, it can initiate synthesis anywhere on a DNA template in vitro. The σ factor is responsible for decreasing the nonspecific binding affinity of the RNA polymerase. The sequence, structure, and function are evolutionarily conserved from bacteria to humans.

12 Structure of bacterial RNA polymerase Sigma factor In bacteria there are many different σ factors. In E. coli the most abundant σ factor is σ 70. It has a higher binding affinity for the RNA polymerase core enzyme than other σ factors. Most of the σ factors share four regions of amino acid sequence homology that play a role in recognizing the promoter These four regions are further divided into subdomains with specific functions. In the holoenzyme the globular domain of the σ factors are spread out across the face of the crab claw.

13 Structure of bacterial RNA polymerase Sigma factor σ 70 is required for specific binding of RNA polymerase to the promoter of the majority of genes in E. coli. It stimulates a change in shape of the internal channel where transcription takes place. The 35 and the 10 sequences are necessary for recognition by the σ 70 factor The 10 sequence is the region of contact for the core enzyme. In addition, the 10 sequence is necessary for the initial melting of the DNA to expose the template strand. A domain of the σ 70 factor binds to the nontemplate strand of the 10 sequence in a sequence-specific manner that stabilizes the initial transcription bubble.

14 The sigma factor, σ 70 (MW = 72000) Fragments 2.1 and 2.2of σ 70 bind strongly to β'. Adjacent helical segments located in fragments 2.3 and 2.4are involved in recognition of the -10 regionof the promoter. The 2.3 region is required for melting. 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 σto the polymerase core gives the RNA polymerase holoenzymerecognizing a site at -10to form the closed complex. In the holoenzymeform, an additional DNA binding domain of σ, 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 holoenzymemelts the region -11 to +3in the DNA, giving the open complex, and the bubble is stabilized by the ssdnabinding domain of σat region 2.3. The region 2.5 interactswith dsdnafrom -11 to 17(spacer region). Melting of the transcription bubble admits the template strand to the catalytic site, allowing initiation to proceed.

15 Alternative sigma factors respond to general environmental changes E. Coli sigma factors recognize promoters with different consensus sequences 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.

16 DNAse I Footprinting 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.

17 DNase I footprint performed on an end-labeled DNA fragment FIS Footprint Samples in lanes 2-4 had increasing amounts of the DNA-binding protein (lambda protein cii); lane 1 had no protein.

18 DNA-protein complex Partially DNase I digested DNA is subjected to linear PCR DNase I Products of DNase I digestion are primer extended by linear PCR using a 5 end-labeled oligonucleotide Sequencing gel

19 Gel retardation assay Gel Shift Electro Mobility Shift Assay (EMSA) Band Shift Incubating a purified protein, or a complex mixture of proteins e.g. nuclear or cell extract, with a 32 P end-labelled DNA fragment containing the putative protein binding site (from promoter region). Reaction products are then analysed on a nondenaturing 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. No protein add protein Non-denaturing PAGE * * Free DNA probe Retarded mobility due to protein binding

20 virb virf virg Bound DNA EMSA Free DNA Evaluating the Binding Affinity

21 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

22 The transcription process consists of three stages: Initiation Elongation Termination

23 Initiation

24 Initiation of transcription The RNA polymerase holoenzyme initially binds to the promoter at nucleotide positions 35 and 10 relative to the transcription start site (+1) to form a closed promoter complex. The term closed indicates that the DNA remains doublestranded and the complex is reversible.

25 Initiation of transcription The complex then undergoes a structural transition to the open form in which approximately 18 bp around the transcription start site are melted to expose the template strand of the DNA. Transcription is aided by negative supercoiling of the promoter region of some genes. Formation of the open complex is generally irreversible and transcription initiated in the presence of NTPs. No primer is required for initiation by RNA polymerase.

26 Initiation of transcription During initial transcription, RNA polymerase produces and releases short RNA transcripts of less then ten ribonucleotides (abortive synthesis) before escaping the promoter (promotor clearance). It is not clear why RNA polymerase must undergo this period of abortive initiation before achieving escape, but a region of the σ factor appear to be involved. Infact, in this step there is sequential displacement of some domains of σ that would otherwise act as a barrier to the extension of the nascent RNA as it emerges from the RNA exit channel.

27 Elongation

28 Elongation After about 9-12 nt of RNA have been synthesized, the initiation complex enters the elongation stage. As RNA polymerase moves during elongation, it holds the DNA strands apart, forming a transcription bubble. The moving polymerase protects a footprint of ~30 bp along the DNA against nuclease digestion. Within the transcription bubble, one strand of DNA acts as the template for RNA synthesis by complementary base pairing. Transcription always proceeds in the 5 3 direction.

29 Proofreading E. coli RNA polymerase synthesizes RNA with remarkable fidelity in vivo. Its low error rate may be achieved by two proofreading mechanisms. Pyrophosphorolytic editing The RNA polymerase use its active site in a simple back-reaction, to catalyze the removal of an incorrectly inserted ribonucleotide by reincorporation of Ppi. Hydrolytic editing The polymerase backtracks by one or more nucleotides and cleaves the RNA product, removing the error-containing sequence. Hydrolytic editing is stimulated by Gre factors, which both enhance hydrolytic function and serve as elongation stimulating factor. Gre factors ensure that polymerase elongates efficiently and help overcome arrest in presence of a mismatch.

30 Termination

31 Termination of transcription The RNA polymerase core enzyme moves down the DNA until a stop signal or terminator sequence is reached by the RNA polymerase. There are two types of terminators recognized, Rho-dependent Rho-independent terminators E. coli uses both kinds of transcript terminators.

32 Termination of transcription Rho-independent termination Also called intrinsic terminators because they cause termination of transcription in the absence of any external factors. Terminator is characterized by a consensus sequence that is an inverted repeat. Stem-loop structures can form within the mrna just before the last base transcribed, by the pairing of complementary bases within the inverted repeat. The inverted repeat sequence in the mrna is followed by seven to eight uracilcontaining nucleotides. A hybrid helix of U in the RNA base paired with A in the DNA is less stable than other complementary base pairs. This property, combined with formation of the stem loop in the exit channel of RNA polymerase, is sufficient to cause the enzyme to pause, resulting in transcript release.

33 Termination of transcription Rho-dependent termination Rho-dependent termination is controlled by the ability of the Rho protein to gain access to the mrna. Terminator is an inverted repeat with no simple consensus sequence and no string of Ts in the nontemplate strand. Rho is a ring-shaped, hexameric helicase protein with a distinct RNAbinding domain and an ATP-binding domain. Rho binds specifically to a C-rich site called a Rho utilization (or rut site) at the 5 end of the newly formed RNA, as it emerges from the exit site of RNA polymerase. Temporary release of one subunit of the hexamer allows the 3 segment of the nascent transcript to enter the central channel of the Rho ring.

34 Termination of transcription Rho-dependent termination In an ATP-dependent process, Rho travels along the RNA, chasing the RNA polymerase. When the polymerase stalls at the terminator stemloop structure, Rho catches up and unwinds the weak DNA RNA hybrid. This causes termination of RNA synthesis and release of all the components.

35 Bacterial gene expression