Chapter 4 Gene Expression: Transcription part 1 Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

Gene Expression-The central Dogma: An Overview 1. Francis Crick (1956) named the flow of information from DNA  RNA protein the Central Dogma. 2. Synthesis of an RNA molecule using a DNA template is called transcription. Only one of the DNA strands is transcribed. The enzyme used is RNA polymerase. 3. There are four major types of RNA molecules: a. Messenger RNA (mRNA) encodes the amino acid sequence of a polypeptide. b. Transfer RNA (tRNA) brings amino acids to ribosome during translation. c. Ribosomal RNA (rRNA) combines with proteins to form a ribosome, the catalyst for translation. d. Small nuclear RNA (snRNA) combines with proteins to form complexes used in eukaryotic RNA processing. Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

The Transcription Process (RNA Synthesis) 1. Transcription, or gene expression, is regulated by gene regulatory elements associated with each gene. 2. DNA unwinds in the region next to the gene, due to RNA polymerase in prokaryotes and other proteins in eukaryotes. In both, RNA polymerase catalyzes transcription . Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

Transcription process Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

3. RNA is transcribed 5’-to-3’. The template DNA strand is read 3’-to-5’. Its complementary DNA, the nontemplate strand, has the same polarity as the RNA. 4. RNA polymerization is similar to DNA synthesis except: a. The precursors are NTPs (not dNTPs). b. No primer is needed to initiate synthesis. c. No proofreading occurs. d. Uracil is inserted instead of thymine. Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

Chemical reaction involved in the RNA polymerase-catalyzed synthesis of RNA on a DNA template strand Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

The Transcription Process Initiation of Transcription at Promoters 1. Transcription is divided into three steps for both prokaryotes and eukaryotes. They are initiation, elongation and termination. The process of elongation is highly conserved between prokaryotes and eukaryotes, but initiation and termination are somewhat different. 2. This section is about initiation of transcription in prokaryotes. E. coli is the model organism. 3. A prokaryotic gene is a DNA sequence in the chromosome. The gene has three regions, each with a function in transcription: a. A promoter sequence that attracts RNA polymerase to begin transcription at a site specified by the promoter. b. The transcribed sequence, called the RNA-coding sequence. The sequence of this DNA corresponds with the RNA sequence of the transcript. c. A terminator region downstream of the RNA-coding sequence that specifies where transcription will stop. Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

Promoter, RNA-coding sequence, and terminator regions of a gene Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

8. RNA polymerase holoenzyme binds promoter in two steps: 4. Promoters in E. coli generally involve two DNA sequences, centered at -35 bp and -10 bp upstream from the +1 start site of transcription. 5. The common E. coli promoter that is used for most transcription has these consensus sequences: a. For the -35 region the consensus is 5’-TTGACA-3’. b. For the -10 region (previously known as a Pribnow box), the consensus is 5’-TATAAT-3’. 6. Transcription initiation requires the RNA polymerase holoenzyme to bind to the promoter DNA sequence. Holoenzyme consists of: a. Core enzyme of RNA polymerase, containing four polypeptides (two α, one β and one β’). b. Sigma factor (σ). 7. Sigma factor binds the core enzyme, and confers ability to recognize promoters and initiate RNA synthesis. Without sigma, the core enzyme randomly binds DNA but does not transcribe it efficiently. 8. RNA polymerase holoenzyme binds promoter in two steps: a. First, it loosely binds to the -35 sequence. b. Second, it binds tightly to the -10 sequence, untwisting about 17 bp of DNA at the site, and in position to begin transcription. Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

Action of E. coli RNA polymerase in the initiation and elongation stages of transcription Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

a. σ70 recognizes the sequence TTGACA at -35, and TATAAT at -10. 9 Promoters often deviate from consensus. The associated genes will show different levels of transcription, corresponding with sigma’s ability to recognize their sequences. 10. E. coli has several sigma factors with important roles in gene regulation. Each sigma can bind a molecule of core RNA polymerase and guide its choice of genes to transcribe. 11. Most E. coli genes have a σ70 promoter, and σ70 is usually the most abundant sigma factor in the cell. Other sigma factors may be produced in response to changing conditions. Examples of E. coli sigma factors: a. σ70 recognizes the sequence TTGACA at -35, and TATAAT at -10. b. σ32 recognizes the sequence CCCCC at -39 and TATAAATA at -15. Sigma32 arises in response to heat shock and other forms of stress. c. σ54 recognizes the sequence GTGGC at -26 and TTGCA at -14. Sigma54 arises in the response to heat shock and other forms of stress. d. σ23 recognizes the sequence TATAATA at position -15. Sigma23 is present in cells infected with phage T4. 12. E. coli has additional sigma factors. Other bacterial species also have multiple sigma factors. Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

The Transcription Process Elongation and Termination of an RNA Chain 1. Once initiation is completed, RNA synthesis begins. After 8–9 NTPs have been joined in the growing RNA chain, sigma factor is released and reused for other initiations. Core enzyme completes the transcript. 2. Core enzyme untwists DNA helix locally, allowing a small region to denature. Newly synthesized RNA forms an RNA-DNA hybrid, but most of the transcript is displaced as the DNA helix reforms. The chain grows at 30–50 nt/second. 3. Terminator sequences are used to end transcription. In prokaryotes there are two types: a. Rho-independent (ρ-independent) or type I terminators have two-fold symmetry that would allow a hairpin loop to form. The palindrome is followed by 4-8U residues in the transcript, and together these sequences cause termination, possibly because rapid hairpin formation destabilizes the RNA-DNA hybrid. b. Rho-dependent (ρ-dependent) or type II terminators lack the poly(U) region, and many also lack the palindrome. The protein ρ is required for termination. It has two domains, one binding RNA and the other binding ATP. ATP hydrolysis provides energy for ρ to move along the transcript and destabilize the RNA-DNA hybrid at the termination region. Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

Sequence of a -independent terminator and structure of the terminated RNA Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

Transcription in Eukaryotes 1.Prokaryotes contain only one RNA polymerase, which transcribes all RNA for the cell. 2. Eukaryotes have three different polymerases, each transcribing a different class of RNA. Processing of transcripts is also more complex in eukaryotes. Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

Eukaryotic RNA Polymerases 1. Eukaryotes contain three different RNA polymerases: a. RNA polymerase I, located in the nucleolus, synthesizes three of the four rRNAs found in ribosomes: three of the RNAs (the 28S, 18S, and 5.8S rRNA molecules). b. RNA polymerase II, located in the nucleoplasm, synthesizes messenger RNAs (mRNAs; translated to produce polypeptides) and some small nuclear RNAs (snRNAs), some of which are involved in RNA processing events. c. RNA polymerase III, also located in the nucleoplasm, synthesizes the transfer RNAs (tRNAs), which bring amino acids to the ribosome; 5S rRNA, the fourth rRNA molecule found in each ribosome; and the small nuclear RNAs (snRNAs) not made by RNA polymerase II. 2. Eukaryotic RNA polymerases are harder to study than the prokaryotic counterpart, because they are present at low concentrations. Inhibition by α-amanitin is a useful research tool, since RNA pol II is very sensitive, RNA pol III less so and RNA pol I is relatively insensitive Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

Transcription of Protein-Coding Genes by RNA Polymerase II 1. When protein-coding genes are first transcribed by RNA pol II, the product is a precursor-mRNA (pre-mRNA). The pre-mRNA will be modified to produce a mature mRNA. 2. Promoters for protein-coding genes are analyzed in two ways: a. Directed mutation. b. Comparison of sequences from known genes. 3. Results of promoter analysis reveal two types of elements: a. Basal promoter elements are located near the transcription start site. Examples include: i. The TATA box (aka TATA element or Goldberg-Hogness box) at -25; its full sequence is TATAAAA. This element in local DNA denaturation, and sets the start point for transcription. ii. The initiator element (Inr), a pyramiding-rich sequence near the transcription start site. b. Promoter proximal elements are further upstream from the start site, at positions between -50 and -200. These elements generally function in either orientation. Examples include: i. The CAAT box, located at about -75. ii. The GC box, consensus sequence GGGCGG, located at about -90. Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

a. Each TF works with only one type of RNA polymerase. 4.Various combinations of basal and proximal elements are found near different genes, and no one element is essential for transcription initiation. 5. Basal transcription factors (TFs) are needed for initiation by all 3 RNA polymerases. a. Each TF works with only one type of RNA polymerase. b. TFs are numbered to match their corresponding RNA polymerase, and assigned a letter in the order of their discovery (e.g., TFIID was the fourth TF discovered that works with RNA polymerase II). 6. For protein-coding genes, binding of TFs and RNA pol II occurs in a set order: a. TFIID binds the TATA box, forming an initial committed complex. b. TFIIB binds the TFIID-TATA box complex. c. The TFIID-TATA box plus TFIIB complex recruits RNA polymerase II and TFIIF, producing the minimal transcription initiation complex. d. TFIIE and TFIIH bind, producing the complete transcription initiation complex, or preinitiation complex (PIC). 7. The PIC allows only a low level of transcription. Higher levels are induced by activator factors that bind DNA sequences called enhancers. Interaction between the enhancer-activator factor complex and the PIC stimulates transcription. Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

Assembly of the Transcription Initiation machinery Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4

8. Characteristics of enhancers: a. They are found in single or multiple copies. b. They function in either orientation. c. They function upstream, downstream or within the gene, although they are usually located upstream. d. They may be several kb from the gene they control. 9. Silencers have properties similar to enhancers, except that they decrease transcription. Repressor factors bind to them. Silencers are not as common as enhancers. 10. The interaction of transcription factors binding promoters, enhancers and silencers results in cell- and tissue-specific gene expression. 11. Upstream activator sequences (UASs) in yeast are similar to enhancers, but cannot function when located downstream of the promoter. Prepared by Prof. Sabah Hassan -Coordination by Prof Sabah Linjawi BIO 702 L 4