Genetic Information Flow: Transcription of Class I, II and III genes Dr. Umut Fahrioglu, PhD MSc

Basics of Transcription Associated with each gene there are sequences called gene regulatory elements. These elements are involved in regulating transcription. The process by which RNA molecules are synthesized from a DNA template is called transcription. This process is catalyzed by the enzyme RNA polymerase (also knows as the DNA-dependent RNA polymerase). RNA is synthesized in the 5’-to-3’ direction. The 3’-to-5’ DNA strand that is read to make the RNA strand is called the template strand. The 5’-to-3’ DNA strand complementary to the template strand and having the same polarity as the resulting RNA strand is called the non-template strand. The polymerization is similar to DNA synthesis, except that no primer is required. The sequences that is represented in databases and literature is usually of the non-template strand.

Sense (non-template or coding) vs Sense (non-template or coding) vs. anti-sense (template or non-coding) strand Sense (non-template or coding) anti-sense (template or non-coding) strand

Bacterial RNA polymerase Bacterial RNA polymerase has been extensively characterized and is shown to consist of α, β, β’, ω and σ. The active form of the enzyme is called the holoenzyme and contains the subunits α2ββ’σ. The holoenzyme is required for the binding and initiation step. It is the σ factor that plays the main function in initiation of transcription at the right place. The other form of the enzyme, known as the core enzyme, α2ββ’ ,can catalyze polymerization but cannot initiate transcription. ω is known to promote assembly of the enzyme. There can be different σ’s creating different holoenzymes. (E. coli has 6 and B. subtilis has 9 different σ’s). Different σ’s recognize different categories of gene. There are also two other factors ρ and NusA that act as termination factors for the E. coli polymerase.

Stages of Transcription A single transcriptional unit is defined as the segment of DNA that is transcribed as a single continuous RNA molecule. The process is complicated and is divided into four stages: Binding Initiation Elongation Termination

The Binding Stage and the Promoter This stage begins with the attachment of polymerase holoenzyme to the DNA at a specific sequence called the promoter. Each transcription unit has a promoter near it. The promoter determines where the RNA synthesis starts and which DNA strand is used as a template. The promoter is upstream of the start site which is 5’ to the start site on the coding strand. The 40 bp or so DNA segment that makes up the promoter differs among transcription units. The point where transcription will begin is always a purine and often an adenine and is called the startpoint. Approximately, 10 bases upstream of the startpoint is the 6-base sequence TATAAT called the -10 sequence or the Prinbow box. Near the -35 position is the 6-base sequence TATGAT called the -35 sequence. Both are conserved in evolution but they are not the same in all prokaryotes and even change from gene to gene in the same genome.

Typical Prokaryotic Promoter

Polymerase and Scanning and some facts Polymerase scans DNA linearly without opening it up. Detects the -10 and -35 sequences at the major grove and the sigma factor allows the core enzyme to bind to the promoter. The separation sequence: the region between the –35 and –10; 16-19 bp and the distance is more important than the sequence No proofreading nuclease activities 1 error in 105 bp RNA polymerase unwinds DNA approximately two turns (17 bp) Elongation rate is 50 nt/sec

The Initiation Stage Once the RNA polymerase is bound to the promoter and the DNA double helix is unwound, the initiation of RNA synthesis is possible. The promoter sequence will determine the orientation of the RNA polymerase which in return will determine which DNA strand is transcribed. As soon as the first two NTPs are hydrogen-bonded with the DNA startpoint, RNA polymerase catalyzes the formation of the phosphodiester bond between the 3’-OH of the first NTP and the 5’-phosphate of the second. The polymerase stays at the promoter until the chain reaches about 9 nucleotides through sequential one by one addition. At this point the sigma factor detaches from the polymerase and the initiation is complete.

Both strands encode genes

The Elongation Stage Chain elongation occurs as the polymerase moves along the DNA molecule. It untwists the helix bit by bit and adds NTPs one by one. The polymerase moves along the DNA template from the 3’ to the 5’ end. The RNA strand is elongated in the 5’ to 3’ direction. Each new nucleotide is added to the 3’ end of the growing chain. The most recently added nucleotides remain base- paired with the DNA strand forming a short RNA-DNA hybrid, which is thought to be about 12 bp long. The polymerase continues to move along, rewinding the DNA behind it and unwinding the DNA ahead of it.

The Termination Stage The termination of transcription takes place after the polymerase has transcribed a special sequence on the DNA called the terminator. It is the RNA transcript of this signal that is the actual termination signal. In prokaryotes, there is usually an RNA sequence that gives rise to a hairpin structure that is followed by a stretch of Us. The hairpin must be rich in GC base pairs. The exact sequence of it is not that important. It is the stretch of Us that cause the polymerase to disassociate from the DNA. This only happens on some transcripts. On other transcripts, the transcription unit needs a protein called the ρ factor. This factor recognizes an RNA sequence 50-90 bases long preceding the termination point. ρ factor binds the transcript as a homohexamer. After termination, the core polymerase is ready to bind another sigma factor again and reinitiate synthesis on another promoter.

Termination-Rho Dependent Terminator RNA Pol. 5’ r Help, rho hit me! RNA Pol. 5’ r Rho catches up with RNA polymerase

Play by play summary of transcription RNA polymerase binds to the -35 region. This is called a closed complex because the DNA is still base-paired. RNA polymerase unwinds DNA approximately two turns (17 bp). This creates what is called a transcription bubble. It is easy to melt the AT rich DNA. RNA polymerase bind to -10 region and bends and opens the DNA. Polymerase binds to the single-stranded DNA. This is called an open complex. As RNA polymerase transcribes DNA, unwinding and rewinding occur. Gyrase and topoisomerase I rectify the supercoiling. First phosphodiester bond is formed. Sigma factor dissociates. Elongation factor binds and elongates at a rate of 50 nt/sec. Rho independent or rho dependent termination

Transcription in Eukaryotes The four stages are the same in eukaryotes but the process is a bit more complex compared to the prokaryotes. There are three different RNA polymerases that transcribe the RNA of the nuclear DNA. Each of them synthesizes one or more particular kinds of RNA. The eukaryotic promoters are more complex and varied compared to the prokaryotic promoter. There is a great variability especially among the ones for the protein coding genes. What makes things more complex is that some promoters are located downstream from the transcription startpoint. Many transcription factors are involved in the binding of the RNA polymerase to the DNA. Unlike the sigma factor, they are not considered part of the polymerase. They must bind to the polymerase before the polymerase binds to the DNA.

Transcription in Eukaryotes cont... Chromatin remodelling is important for the initiation of transcription. Cis –acting upstream DNA elements are important for regulation and initiation. Enhancers can be found both upstream and downstream. Protein-protein interactions are very important in the first stage of eukaryotic transcription. (Trans-acting protein factors) The RNA cleavage at the 3’ end is more important than the termination itself. Extensive RNA processing takes place during and after transcription in eukaryotes.

Eukaryotic RNA Polymerases The nuclear enzymes are classified as RNA polymerase I, II and III. Each contains 10 or more peptides. None can bind to a promoter by itself. They are structurally similar to each other and to the prokaryotic core RNA polymerase. RNA polymerase I: Found in the nucleolus. Responsible for the synthesis of the RNA molecule that is the precursor to the 3 of the 4 RNA molecules of the ribosome. Accounts for most of the cellular RNA synthesis. RNA polymerase II: Found in the nucleoplasm. Responsible for the synthesis of the precursor to the mRNA. In addition, it synthesizes snRNA, which are small RNA that take part in posttranscriptional RNA processing. Responsible for the greatest variety of RNA molecules. RNA polymerase III: Also found in the nucleoplasm. Responsible for the synthesis of a variety of small RNAs, that include tRNA and the smallest piece of rRNA

Summary of Eukaryotic RNA polymerases

Eukaryotic Promoters: For RNA Polymerase I Even though they are very varied they can still be classified into three main types, one for each type of polymerase. The promoter for polymerase I has two parts. The core promoter that extends into the nucleotide sequence to be transcribed. However, the transcript is made much more efficient by the presence of the upstream control element, which is a fairly long sequence in this case that looks like the core promoter. The binding of the transcription factors to both of these promoters facilitate the binding of the RNA polymerase I to the core polymerase and enable it to initiate transcription.

Eukaryotic Promoters: For RNA Polymerase II Core promoter is the set of cis-acting sequence elements needed for the transcription machinery to start transcription at the correct site. A typical promoter for RNA polymerase II has a short initiator (Inr) sequence surrounding the startpoint. Around -25, a short sequence called the TATA box, which has the consensus sequence TATA followed by two or more As. (Goldberg- Hogness box) B-recognition element (BRE) consists of 7 nucleotides (G/C G/C G/A C G C C) and is found upstream of the TATA element. Downstream promoter element (DPE) a distinct 7-nucleotide core promoter element that is ∼30 nucleotides downstream of the transcription start site The core promoter is only a basal level promoter. The Inr and the TATA box only specify where the transcription machinery will assemble and determine where the transcription will begin. Most promoters will have upstream control elements to improve the promoters efficiency.

Proximal Control Elements and Enhancers The proximal control elements are upstream from the TATA box, around the area of 50 to 200 nucleotides from the transcription start site. These elements are important in determining when and how a gene is expressed. Key to this regulation is transcription regulatory proteins called activators. Housekeeping genes like actin, glucose 6-phosphate dehydrogenase have proximal control elements that are recognized by activators found in all cell types. CAAT box named for its consensus sequence is located -75 GC box with consensus sequence 5’-GGGCGG-3’ located at about -90 Both of these elements work in either direction and a mutation in either one of them markedly decrease transcription initiation. Other sequences called enhancers are required for the maximal transcription of a gene. Enhancers function either upstream or downstream from the transcription initiation site. They are sometimes thousands of base pairs away. They modulate transcription from a distance.

Transcription factors Complementing the cis-acting regulatory sequences there are trans-acting factors that facilitate RNA polymerase II binding and initiation of transcription. These factors are called transcription factors. We can divide them into two broad categories: General transcription factors: These are the factors that are absolutely required for the RNA pol II mediated transcription. They are well characterized and include TFIIA, TFIIB and so on. TFIID binds to the TATA box. TFIID has 10 subunits and one of them is called the TATA-binding protein (TBP). Once the initial binding occurs, there are many other subunits that bind to this factor and form an extensive pre initiation complex. The GC box is bound by SP1 factor (affects the frequency of initiation) Specific transcription factors: These effect the efficiency and rate of RNA pol II. Different genes have many different specific transcription factors that may only be present at a specific time in the cell’s life.

Promoter (DNA sequence upstream of a gene) transcription unit exon promoter P TE transcription element Promoter (DNA sequence upstream of a gene) determines start site (+1) for transcription initiation located immediately upstream of the start site allows basal (low level) transcription Transcription element (DNA sequence that regulates the gene) determines frequency or efficiency of transcription located upstream, downstream, or within genes can be very close to or thousands of base pairs from a gene includes enhancers (increase transcription rate) silencers (decrease transcription rate) response elements (target sequences for signaling molecules) genes can have numerous transcription elements +1

exon P TE transcription unit promoter transcription element promoter complex

Sequence elements within a typical eukaryotic gene1 GC TATA CAAT -25 -50 -80 -95 -130 1 based on the thymidine kinase gene octamer transcription element promoter TATA box (TATAAAA) located approximately 25-30 bp upstream of the +1 start site determines the exact start site (not in all promoters) binds the TATA binding protein (TBP) which is a subunit of TFIID GC box (CCGCCC) binds Sp1 (Specificity factor 1) CAAT box (GGCCAATCT) binds CTF (CAAT box transcription factor) Octamer (ATTTGCAT) binds OTF (Octamer transcription factor) +1 ATTTGCAT

Eukaryotic Promoters: RNA polymerase III These promoters vary in structure but the ones for tRNA and 5S rRNA are located entirely downstream of the transcription startpoint, within the transcribed gene. Boxes A, B and C are DNA sequences each about 10bp long. In tRNA genes, about 30 to 60 bp of DNA separate the boxes A and B. In 5S rRNA , about 10 to 30 bp separate the boxes A and C.

Sequence of events for mRNA synthesis TFIID (a multisubunit protein) binds to the TATA box to begin the assembly of the transcription apparatus The TATA binding protein (TBP) is one of the subunit of TFIID that directly binds the TATA box. Binds 105-fold higher to TATA box than to random DNA sequence TBP associated factors (TAFs) bind to TBP. TFIIA, TFIIB, TFIIE, TFIIF, TFIIH, TFIIJ assemble with TFIID. TFIIH is also involved in phosphorylation of RNA polymerase II, DNA repair (Cockayne syndrome mutations), and cell cycle regulation. RNA polymerase II binds the promoter region via the TFII’s. Transcription factors binding to other promoter elements and transcription elements interact with proteins at the promoter and further stabilize (or inhibit) formation of a functional pre-initiation complex.

Formation of a stable pre-initiation complex +1 TBP TFIID B E F H J RNA pol II the stability and frequency with which complexes are formed determines the rate of initiation of transcription the rate of initiation of transcription is of major importance in determining the abundance of an mRNA species TFs recruit histone acetylase to the promoter

Binding of specialized TFs +1 TBP TFIID A B E F H J RNA pol II transcription factors binding to other promoter elements and transcription elements interact with proteins at the promoter and further stabilize (or inhibit) formation of a functional preinitiation complex this process is called “transactivation”

Quick glance at the eukaryotic mRNA RNA processing achieves three things: Removal of introns Addition of a 5’ cap Addition of a 3’ tail This signals that the mRNA is ready to move out of the nucleus and may control the mRNA’s lifespan in the cytoplasm