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Chapter 5 Gene Expression: Transcription The flow of information from DNA RNA protein was called the Central Dogma by Francis Crick in 1956. Synthesis of an RNA molecule using a DNA template is called transcription. The process of coding the RNA information into proteins is called translation. Transcription Only one of the DNA strands is transcribed. The enzyme used is RNA polymerase. 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 ribosome, the catalyst for translation. proteins to form a d. Small nuclear RNA (snRNA) combines with proteins to form complexes used in eukaryotic RNA processing 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 (Figure 5.1). 3. RNA is transcribed 5’-to-3’. The template DNA strand is read 3’-to-5’. Its complementary DNA, the non-template strand, has the same polarity as the RNA. 4. RNA polymerization is similar to DNA synthesis (Figure 5.2), except: a. The precursors are rNTPs (not dNTPs). b. No primer is needed to initiate synthesis. c. Uracil is inserted instead of thymine. d. Only one strand is copied (coding strand 3’to5’) Transcription, the process Transcription is divided into three steps for both prokaryotes and eukaryotes. They are: Initiation Elongation Termination. The process of elongation is highly conserved between prokaryotes and eukaryotes, but initiation and termination are somewhat different. Transcription in prokaryotes E. coli is the model organism (Figure 5.3). A prokaryotic gene is a DNA sequence in the chromosome. This gene has three regions. Each of these regions has a function in transcription: a. The promoter: A specific sequence that attracts the RNA polymerase to begin transcription at a specific location. b. RNA-coding sequence. This is the DNA information that is going to be transcribed into mRNA. c. A terminator region that specifies where transcription will stop. The RNA polymerase RNA polymerase is a holoenzyme. Holoenzymes are enzymes that require a cofactor binding to the Protein portion for the enzyme to be active. The protein portion is called “the apoenzyme”. The RNA polymerase comprises of a. A core enzyme, containing four polypeptides (two α, one β, and one β’). b. Sigma factor (σ). Sigma factor binds the core enzyme, and confers ability to recognize promoters. The Promoter The presence of a promoter sequence determines which strand of the DNA helix is the template and inside the promoter we find the starting point for the transcription of a gene. The promoter also includes a binding site for RNA polymerase (several dozen nucleotides upstream of the start point). The enzyme recognizes this binding site and binds directly to the promoter region. Promoters in E. coli generally involve two DNA sequences, centered at -35bp and -10bp upstream from the +1 start site of transcription. Very commonly, not always, for the -35 region the consensus is 5’-TTGACA-3’ and for the -10 region (previously known as a Pribnow box), the consensus is 5’-TATAAT-3’. Initiation Transcription initiation requires the RNA polymerase holoenzyme to bind to the promoter DNA sequence . It takes places in two steps: a. First, it loosely binds to the -35 sequence of dsDNA (closed promoter complex). b. Second, it binds tightly to the -10 sequence, untwisting about 17bp of DNA at the site, and in position to begin transcription (open promoter complex). At this point the RNA polymerase is correctly oriented to begin transcription at the +1 nucleotide. After about 10 bases have been polymerized (transcribed) the sigma factor dissociates from the enzyme. The associated genes will show different levels of transcription, corresponding with sigma’s ability to recognize their sequences. E. coli has several different sigma factors with important roles in gene regulation. Other bacterial species also have multiple sigma factors. For example, most E. coli genes have a σ70 promoter, and σ70 is usually the most abundant sigma factor in the cell. However, other sigma factors may be produced in response to changing conditions. Examples of E. coli sigma factors are: σ32, σ54 and σ23 Elongation of an RNA Chain 1. Once initiation is completed, RNA synthesis begins. After 8–9 rNTPs have been joined in the growing RNA chain, sigma factor is released and reused for other initiations. Core enzyme completes the transcript (Figure 5.4). 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– 50nt/second. 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. Termination Terminator sequences are used to end transcription. In prokaryotes there are two types: a. Rho-independent (r-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 4–8 U residues in the transcript, and when these sequences are transcribed, they cause termination. Rho-dependent (r-dependent) or type II terminators lack the poly(U) region, and many also lack the palindrome. The protein rho is required for termination. It requires energy. Transcription in Eukaryotes Prokaryotes contain only one RNA polymerase, which transcribes all RNA for the cell. Eukaryotes have three different polymerases, each transcribing a different class of RNA. Processing of transcripts is also more complex in eukaryotes. Eukaryotic RNA Polymerases 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. All known eukaryotic RNA polymerases have multiple subunits Initiation of transcription. The transcription initiation complex is composed of promoter sequences and DNA binding proteins. These two components of transcription are normally described as cis-acting elements (promoter sequences) and trans-acting factors (binding proteins) Cis-acting elements are- DNA sequences in the vicinity of the structural portion of a gene that are required for gene expression. The promoter of protein coding genes in eukaryote cells are located about 200 base pairs upstream of the transcription initiation site and contains various elements. We can divide the promoter into two areas, the core promoter and the promoter proximal elements. The core promoter is usually a DNA sequence located about -50 from the transcription starting point. This sequence is divided in sections or “elements”. The best known elements are the INR, initiator and the TATA box(GoldbergHogness box). The core promoter determines where the transcription will begin. The promoter proximal elements are further up upstream (from -50 to -200) and the most well known element is the CAAT box located about -75 and the CG box. The promoter proximal elements determine when and how the gene is going to be expressed. Activators: Proteins that regulate transcription. Enhancers: Required for maximal transcription of a gene. They can be upstreqam or downstream. Transcription initiation factors. GTF’ that are numbered and 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). Sequence of binding is not completely understood. Eukaryotic mRNAs 1. Eukaryotic mRNAs have three main parts (Figure 5.8): a. The 5’ leader sequence, or 5’ untranslated region (5’ UTR), varies in length. b. The coding sequence, which specifies the amino acid sequence of the protein that will be produced during translation. It varies in length according to the size of the protein that it encodes. c. The trailer sequence, or 3’ untranslated region (3’ UTR), also varies in length and contains information influencing the stability of the mRNA. Eukaryotes modify pre-RNA into mRNA by RNA processing and then the processed mRNA migrates from nucleus to cytoplasm before translation. Eukaryotic pre-RNAs often have introns (intervening sequences) between the exons (expressed sequences) that are removed during RNA processing. Introns were discovered in 1977 by Richard Roberts, Philip Sharp, and Susan Berger. 5’ Modifications The newly made 5’ end of the mRNA is modified by 5’ capping. The 5' cap is modified guanine nucleotide (methylated and known as a 7-methyl guanosine m7G ) is added to 5‘ using a 5’-to-5’ linkage. Sugars of the two adjacent nt are also methylated. This modification is critical for recognition and proper ribosome binding to the mRNA during translation initiation (Figure 5.10). It may also be important for other essential processes, such as splicing and transport. The 3’ end of mRNA is marked by a poly(A) tail (Figure 5.11). a. Transcription of mRNA continues through the poly(A) sequence (AAUAAA). b. Proteins bind and cleave RNA. c. After cleavage, the enzyme poly(A) polymerase (PAP) adds A nucleotides to the 3’ end of the RNA, using ATP as a substrate. d. PABII (poly(A) binding protein II) binds the poly(A) tail as it is produced. RNA splicing Introns typically begin with 5’-GU, and end with AG3’, but more than just these sequences is needed to specify a junction between exon and intron. The removal of the introns is called RNA splicing and it involves a complex called spliceosomes. A spliceosome consists of the preRNA and a group of proteins; the small nuclear ribonucleoprotein particles (snRNPs) also known as snurps. There are labeled U1, U2, U3, U4, U5 and U6. Transcription on non-protein coding genes. tRNA and rRNA’s do not code for proteins, nevertheless their sequence needs to be transcribed for their existence. Ribosomes are the catalyst for protein synthesis, facilitating binding of charged tRNAs to the mRNA so that peptide bonds can form. A cell contains thousands of ribosomes. Prokaryote Ribosome The prokaryotic ribosome is a 70S ribosome, with two subunits : A large, 50S, and the small, 30S. subunits (Figure 5.16). The 50S subunit consists of two molecules of rRNA, the 23S rRNA (2,904nt) and 5S rRNA (120nt), plus 34 different proteins. The 30S subunit contains only one rRNA molecule, the 16S rRNA (1,542nt), plus 20 different proteins. S or Sverberg is a unit of centrifugation The eukaryotic ribosome Eukaryotic ribosomes are larger and more complex than prokaryotic ones, and vary in size and composition among organisms. Mammalian ribosomes are an example; they are 80S, with 60S and 40S subunits (Figure 5.17). The 60S subunit contains the 28S rRNA (~4,700nt), the 5.8S rRNA (156nt), and the 5S rRNA (120nt), plus about 50 proteins. The 40S subunit contains the 18S rRNA (~1,900nt) plus about 35 proteins. Transcription of rRNA Genes DNA regions that encode rRNA are called ribosomal DNA (rDNA) or rRNA transcription units (genes?). E. coli is a typical prokaryote, with seven rRNA coding regions, designated rrn, scattered in its chromosome (Figure 5.18). Each rrn contains the rRNA genes 16S-23S-5S, in that order, with tRNA sequences in the spacers. A single pre-rRNA transcript is produced from rrn, and cleaved by RNases to release the rRNAs. Cleavage occurs in a complex of rRNA and ribosomal proteins, resulting in functional ribosomal subunits.