MB 6 Transcription in Prokaryotes and Eukaryotes PDF

Transcription in Prokaryotes and Eukaryotes  Gene Expression: 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 tr...

Transcription in Prokaryotes and Eukaryotes  Gene Expression: 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 ribosomes 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.  The Transcription Process RNA Synthesis RNA Biosynthesis 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. The Transcription Process  The Transcription Process 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. d. Uracil is inserted instead of thymine. Promoter, RNA-coding sequence, and terminator regions of a gene  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. (Figure 5.3) 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.  Promoter, RNA-coding sequence, and terminator regions of a gene 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).  Promoter, RNA-coding sequence, and terminator regions of a gene 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 (Figure 5.4): 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. Action of E. coli RNA polymerase in the initiation and elongation stages of transcription  Promoter, RNA-coding sequence, and terminator regions of a gene 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.  Promoter, RNA-coding sequence, and terminator regions of a gene 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.  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. RNA polymerase has two types of proofreading: a. Similar to DNA polymerase editing, newly inserted nucleotide is removed by reversing synthesis reaction. b.Enzyme moves back one or more nucleotides, cleaves RNA, then resumes synthesis in forward direction.  The Transcription Process Elongation and Termination of an RNA Chain 4. 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 (Figure 5.5). The palindrome is followed by 4- 8U residues in the trasncript, 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 destablize the RNA-DNA hybrid at the termination region Sequence of a -independent terminator and structure of the terminated RNA  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.  Eukaryotic RNA Polymerases 1. Eukaryotes contain three different RNA polymerases: a. RNA polymerase I, located in the nucleolus, transcribes the three major rRNAs (28S, 18S, and 5.8S). b. RNA polymerase II, located in the nucleoplasm, transcribes mRNAs and some snRNAs. c. RNA polymerase III, located in the nucleoplasm, transcribes tRNAs, 5S rRNA, and the remaining snRNAs. 2. Eukaryotic RNA polymerases are harder to study than the prokaryotic counterpart, because they are present at low concentrations. 3. All known eukaryotic RNA polymerases have multiple subunits. An example is yeast RNA pol II with 12 subunits, 5 of which are also in its RNA pol III  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. Promoter analysis reveal two types of elements: a. Core promoter elements are located near the transcription start site and specify where transcription begins. Examples include: i. The initiator element (Inr), a pyramidine-rich sequence that spans the transcription start site. Transcription of Protein-Coding Genes by RNA Polymerase II ii. The TATA box (also known as a TATA element or Goldberg-Hogness box) at -30; its full sequence is TATAAAA. This element aids in local DNA denaturation, and sets the start point for transcription. b.Promoter proximal elements are required for high levels of transcription. They 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.  Transcription of Protein-Coding Genes by RNA Polymerase II 4. Various combinations of core and proximal elements are found near different genes. Promoter proximal elements are key to gene expression. a. Activators, proteins important in transcription regulation, are recognized by promoter proximal elements. b.Housekeeping (used in all cell types for basic cellular functions) genes have common promoter proximal elements and are recognized by activator proteins found in all cells. Examples: i. Actin c. Genes expressed only in some cell types or at particular times have promoter proximal elements recognized by activator proteins found only in specific cell types or times.  Transcription of Protein-Coding Genes by RNA Polymerase II 5. Enhancers are another cis-acting element. They are required for maximal transcription of a gene. a. Enhancers are usually upstream of the transcription initiation site, but may also be downstream. They may modulate from a distance of thousands of base pairs away from the initiation site. b.Enhancers contain short sequence elements, some similar to promoter sequences. c. Activators bind these sequences and other protein complexes form, bringing the enhancer complex close to the promoter and increasing transcription. Assembly of the transcription initiation machinery  Transcription of Protein-Coding Genes by RNA Polymerase II 6.Transcription initiation requires assembly of RNA polymerase II and binding of general transcription factors (GTFs) on the core promoter. a.GTFs are needed for initiation by all three RNA polymerases. b. GTFs are numbered to match their corresponding RNA polymerase, and lettered in the order of discovery (e.g., TFIID was the fourth GTF discovered that works with RNA polymerase II).  Transcription of Protein-Coding Genes by RNA Polymerase II 7. Sequence of binding is not completely understood. a. Binding of GTFs and RNA pol II occurs in a set order in in vitro experiments (Figure 5.7) to produce the complete transcription initiation complex or preinitiation complex (PIC): b.The situation is less clear in vivo. Some data indicate that initiation complex forms before binding promoter. c. Transcription for eukaryotes is also complicated by the nucleosome organization of chromosomes. Eukaryotic Transcription (DNA→ RNA) is more complex, slower and more flexible than in Prokaryotes: Gross differences in prokaryotic and eukaryotic transcription. 5 Eukaryotic RNA Polymerase subtypes Core promoters on eukaryotic DNA Transcription factors are required for RNAPolymerase-DNA binding/transcription Relative RNA content in eukaryotic cells Eukaryotic Transcription (DNA→ RNA) is more complex, slower and more flexible than in Prokaryotes: Relative RNA content in eukaryotic cells rRNA production by Polymerase I and processing mRNA production by RNApolymerase II and processing tRNA production by RNApolymerase III and processing Removal of introns from pre-mRNA in the nucleus Eukaryotes have 5 different RNA polymerases Each subtypes makes a specific type of RNA 1) RNAP I: makes rRNA in the nucleolus (darkest part) 2) RNAP II: makes mRNA and snRNA in nucleoplasm 3) RNAP III: makes rRNA and tRNA in nucleoplasm RNAPolymerases are massive complexes: 500,000mw Similar to Prokaryotic RNAPolymerases Eukaryotes have 5 different RNA polymerases Each subtypes makes a specific type of RNA 4) Mitochondria: has mRNAP 5) Chloroplast: has cRNAP How much of each type of RNA are contained in a cell? rRNA: about 75% tRNA: about 15% mRNA: less than 10% Relative to prokaryotes, eukaryotes have much greater flexibility in terms of their ability to modify protein production. This comes at the cost of greater complexity and time needed to make a protein.