Authored by Peter J. Russell CHAPTER 5 Gene

Authored by Peter J. Russell CHAPTER 5 Gene

Authored by Peter J. Russell CHAPTER 5 Gene Expression: Transcription Gene Expression an Overview All life processes depend on the production of gene products present in the cells and tissues Synthesis of RNA using a DNA template is termed transcription Only one of the DNA strands (template strand) is transcribed, employing RNA polymerase Information flow from DNA RNA Protein is called the Central Dogma of

Molecular Biology There are four major types of RNA molecules: Messenger RNA (mRNA) encodes the amino acid sequence of a polypeptide Transfer RNA (tRNA) shuttles amino acids to ribosomes during the translation of mRNA (protein synthesis) Ribosomal RNA (rRNA) combines with proteins to form a ribosome, the catalyst for translation Small nuclear RNA (snRNA) combines with proteins to form complexes used in eukaryotic RNA processing

Transcription Process Transcription is controlled by regulatory elements associated with each gene The unwinding of DNA is accomplished with the help of RNA polymerase in prokaryotes and by a combination of RNA polymerase and other proteins in eukaryotes Transcription proceeds in the 5 3 direction and is catalyzed by RNA pol RNA polymerization is similar to DNA synthesis, with some notable exceptions

DNA replication takes place only during S phase; transcription of RNA occurs throughout the cell cycle Precursors are NTPs (not dNTPs) No primer is needed to initiate synthesis Uracil is inserted instead of thymine Animation: RNA Biosyntheis Summary: RNA Transcription v. DNA replication DNA RNA replication transcripti on Make what from what?

DNA from DNA RNA from DNA # template strands 2 1 Primer needed? Yes No

Proofreading Yes ? Yes Precursors dNTPs (GATC) NTPs (GAUC) Main enzyme DNA Polymerase RNA Transcription in Prokaryotes

Transcription is divided into 3 steps: initiation, elongation and termination Whereas elongation is highly conserved, initiation and termination are somewhat different between eukaryotes and prokaryotes (we will discuss prokaryotes here) A typical prokaryotic gene has three regions: A Promoter sequence attracts the RNA polymerase to begin transcription The transcribed sequence is the coding sequence that is transcribed to make RNA

The terminator region specifies where transcription ends Initiation of Transcription in E. coli The initiation of transcription requires that the RNA polymerase holoenzyme (only one type in bacteria) bind to the Promoter DNA

sequence Promoters are found at -35 and -10 bp upstream from the +1 start site of transcription; their general consensus sequences are: o -35 bp consenus sequence is 5-TTGACA-3 o -10 bp consensus sequence is 5-TATAAT-3 (Pribnow box) Promoters often deviate from their consensus (in a moment) The RNA polymerase holoenzyme is made up of a core enzyme (2 , 1 and polypeptides) bound to a (sigma) factor The factor confers promoter-specific binding (DNA sequence) to the RNA polymerase, otherwise RNA polymerase would bind to nonspecific sites throughout the DNA E. coli has more than 1 sigma factor, which can be produced in response to changing conditions, thereby regulating which promoter is recognized and which gene RNA pol will transcribe The most common factor is 70, which recognizes the exact consensus sequences at -35 and -10 bp Activators and Repressors are regulatory proteins that bind near the promoter to further tweek expression (in Chapter 17)

The RNA polymerase holoenzyme binds to the promoter in two steps involving the factor First, it loosely binds to the -35 bp consensus sequence of ds DNA (closed promoter complex) Second, it binds tightly to the -10 bp consensus sequence, untwisting the DNA at the site, beginning transcription (open promoter complex) Elongation of an RNA Chain As transcription proceeds, the factor dissociates from the core enzyme after about 8 nucleotides are added (note the compaction of RNA pol)

As the RNA polymerase core enzyme continues to add NTPs to the growing RNA strand, the DNA helix reforms, displacing the RNA transcript The transcription bubble moves downstream across the gene at about 30-50 nucleotides per second Eine kleine aufschreiben (a little note) RNA polymerase has proofreading activity similar to that of DNA polymerase The enzyme moves back one or more nucleotides, excises the errant RNA and then resumes synthesis in the forward direction (termed 3 5 exonuclease activity) Termination in Prokaryotes

Terminator sequences are used to end transcription In prokaryotes there are two types of terminator sequences and two mechanisms involved in termination: Rho-independent (r-independent or type I terminators) Rho-dependent (r-dependent or type II terminators) Rho-independent termination

Rho-independent (r-independent) or type I terminators (DNA) consist of an inverted C/G rich repeat sequence that is about 20 bp upstream of the transcription termination point (A rich) RNA pol transcribes the terminator sequence, which is part of the initial RNAcoding sequence of the gene Because of the inverted repeat arrangement (twofold symmetry), the RNA forms a hairpin loop, casing RNA pol to slow its catalysis of synthesis. The string of U nucleotides downstream of the hairpin destabilizes the DNA-RNA hybrid, facilitating the release of RNA transcript and RNA polymerase Rho-Dependent Termination

Rho-dependent (r-dependent) or type II terminators are C-rich, G-poor sequences that have no hairpin structure and require the rho factor protein for termination Rho binds to the C-rich terminator sequence in the RNA transcript (which is upstream of the termination site) and moves along the transcript until encountering the stalled RNA polymerase (which is downstream of Rho) It then acts as a helicase, destabilizing the RNA-DNA hybrid at the termination region, thereby terminating transcription This process is dependent on ATP hydrolysis Transcription in Eukaryotes

Prokaryotes contain only one RNA polymerase, which transcribes all types of RNA for the cell RNA polymerase is multi-subunit protein in prokaryotes and eukaryotes Eukaryotes have three different polymerases, each transcribing a different class of RNA: RNA polymerase I, located in the nucleolus, transcribes the three major rRNAs (28S, 18S, and 5.8S). RNA polymerase II, located in the nucleoplasm, transcribes mRNAs and some snRNAs. RNA polymerase III, located in the nucleoplasm, transcribes the

tRNAs, 5S rRNA and and additional snRNAs R,M,T 1,2,3 Transcription of mRNA by RNA Polymerase II Transcription begins at the promoter; there are two types of promoter elements in eukaryotes: Core promoter elements are located near the transcription start site and specify the origin of transcription. Examples include The initiator element (Inr) spans the transcription start site (+1) The TATA box (Goldberg-Hogness box) aids in local DNA

denaturation. Its full sequence is TATAAAA (-30) Proximal promoter elements are required for high levels of transcription. They are further upstream from the start site (at positions between -50 and -200) The CAAT box is located at about -75 The GC box is located at about -90 (consensus sequence 5GGGCGG-3 ) Activators are recognized by proximal promoter elements and determine the efficiency of transcriptional initiation

Housekeeping genes (used in all cells for basic function) have common proximal promoter elements that are recognized by activator proteins found in all cells Genes expressed only in some cell types or at particular times have proximal promoter elements recognized by activator proteins found only in specific cell types or times Enhancers are another cis-acting element required for the maximal transcription of a gene Enhancers

are usually far upstream (1000s of bp) of the transcription initiation site but may also be downstream Enhancers contain short sequence elements, some similar to promoter sequences Activators bind to these sequences and other protein complexes form, bringing the enhancer complex close to the promoter, thereby increasing transcription Transcription Initiation in Eukaryotes In eukaryotes, RNA polymerases cannot bind to the promoter on its own; it needs the binding of general transcription factors (GTFs) on the core promoter TFIID is a multisubunit protein composed of the TATA-binding

protein (TBP) and TBPassociated factors (TAFs). TFIID binds to the TATA box forming the initial committed complex Sequentially, other factors are recruited, as is RNA polymerase II, to progress through the minimal and complete transcription initiation complexes TFIIH acts like a helicase Structure and Production of Eukaryotic mRNA Mature mRNA has a 5 untranslated region (called the leader sequence), a protein coding sequence and a 3 untranslated region (called the trailer sequence)

Eukaryotes and prokaryotes produce mRNA differently Animation: mRNA Production in Eukaryotes In prokaryotes, polycistronic transcription and translation are coupled processes taking place in the cytoplasm (there are no organelles in prokaryotes)

In eukaryotes, monocistronic transcription and processing of precursor mRNA takes place in the nucleus, producing mature mRNA that is exported to the cytoplasm. The processes of transcription, processing and export are coupled (continuous)

Processing includes: The addition of a 5 cap The addition of a 3 tail The removal of introns. 5 Methyl Guanosine Cap Modification The newly made 5 end of the mRNA is modified by a 5 cap. A capping enzyme adds a guanine, usually 7-methyl guanosine (m7G), to the 5 end using a 5-to-5 linkage. Sugars

of the two adjacent nucleotides are also methylated Functionally, the cap: Protects the mRNA from degradation Facilitates ribosome binding to mRNA during the initiation of translation

3 poly-A Tail Modification ~200 adenine nucleotides are added to the 3 end of mRNA in 2 steps Transcription continues through the poly(A) consensus sequence (AAUAAA). CPSF (cleavage and polyadenylation specificity factor) CstF (cleavage stimulation factor), and CFI and CFII (cleavage factor proteins) bind and cleave the mRNA Poly-A polymerase (PAP) then adds the poly-A tail on mRNA, to which poly-A binding proteins (PABII) attach RNA pol II stops transcription by an unclear mechanism (53 exonuclease on mRNA?) Functionally, the tail:

Protects the mRNA from degradation Facilitates export of the mRNA from the nucleus Introns and splicing in Eukaryotic pre-mRNA Eukaryotic pre-mRNAs have intervening (intron) sequences between the expressed (exon) sequences. Spliceosomes remove the introns during RNA processing Spliceosomes are small nuclear ribonucleoprotein particles (snRNPs are RNA and protein) associated with pre-mRNA Animation: RNA splicing

Spliceosome model summarized... The 5 end of an intron is typically GU; the 3 end is typically AG. Near the end of the intron is an A nucleotide located within the branchpoint sequence, which, in mammals, is YNCURAY, where Y=pyrimidine, N=any base, R=purine, A=adenine. In yeast, the sequence is UACUAAC. (The italicized A in each sequence is where the 5 end of the intron bonds). With the aid of snRNPs, intron removal begins with a cleavage at the first exonintron junction. The G at the released 5 of the intron folds back and forms an unusual 2-5 bond with the A of the branch-point sequence. This reaction produces a lariat-shaped intermediate. Cleavage at the 3 intronexon

junction and ligation of the two exons completes the removal of the intron. so why have introns, you ask? There can be mixing and matching of similar introns between 2 different genes, thereby increasing beneficial crossover events, creating novel genes Alternate splicing of introns takes place in different tissues (cells), producing different gene products and proteins which possess optimal

functions in that tissue (cells) Thus, 1 gene > many polypeptides Alternative Splicing: Many Transcripts from One Gene Self-splicing Introns Most rRNA genes nave no introns Tetrahymena has an intron interrupting its 28S rRNA sequence The intron self-splices by folding into a secondary structure that catalyzes its own excision (no proteins are involved in Group I excision!) Such RNAs with enzyme-like activity are called ribozymes (not classically an enzyme since the catalyst is not in its original form)

Group I introns are also found in nuclear rRNA, mitrochondrial mRNA and tRNA in phages Group II introns use a different molecular mechanism for selfsplicing Lead to RNA world hypothesis of the origin of life RNA editing RNA editing adds or deletes nucleotides from a pre-mRNA, or chemically alters the bases, resulting in an mRNA with bases that dont match its DNA coding sequence. In Trypanosome brucei, the cytochrome oxidase

subunit III gene (from mitochondrial DNA) does not match its mRNA. Uracil residues have been added and removed, and over 50% of the mature mRNA consists of posttranscriptionally added Uracil. Transcription of Other Genes Genes for rRNA, tRNA, and snRNA 1. Ribosomal RNA and Ribosomes: Ribosomes are sites for protein synthesis. Ribosomes are made up of 2 unequal subunits.

Prokaryotic ribosomes are 70S in size: 50s and 30s Eukaryotic ribosomes are 80s in size: 60s and 40s Transcription of rRNA genes DNA regions that encode rRNA are called ribosomal DNA (rDNA) or rRNA transcription units bosome made up of 2 subunits nscription of rRNA genes ranscription of tRNA All tRNAs can be shown in a cloverleaf structure, with complementary base

pairing between regions to form four stems and loops (Figure 5.20). Loop II contains the anticodon used to recognize mRNA codons during translation. Folded tRNAs resemble an upside-down L.

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