Molecular Biology I BIO316 Lecture 7 PDF

Molecular Biology I BIO316 Lecture 7 Summary of transcription among Prokaryotes and Eukaryotes Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics ...

Molecular Biology I BIO316 Lecture 7 Summary of transcription among Prokaryotes and Eukaryotes Prepared by Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  Gene Transcription Associate Professor of Biochemistry and Molecular Genetics  The primary function of genetic material is to store the information necessary for a living organism.  The information is contained within units called genes.  A gene is a segment of DNA that contains the information to make a functional product (RNA molecule or polypeptide).  Protein-coding genes carry the information for the synthesis of polypeptides (amino acid residues).  When a protein-coding gene is transcribed, the first product is called messenger RNA (mRNA).  Gene expression is the overall process by which the information within a gene is used to produce a functional polypeptide.  The promoter provides a site for beginning transcription.  The terminator specifies the end of transcription.   Gene Transcription  The base sequence in the RNA transcript is complementary to the template strand of DNA.  The opposite strand of DNA is the non-template strand.  The non-template strand is also called the coding strand, or sense strand.  This because its sequence is the same as the transcribed mRNA that codes a polypeptide, except that the DNA has thymine (T), where the mRNA contains uracil (U).  The template strand is also called the noncoding strand, or antisense strand.  Transcription factors are a category of proteins that bind to genes and control the rate of transcription.  Some transcription factors bind directly to the promoter and facilitate transcription. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics   Gene Transcription  Other transcription factors recognize regulatory elements (also called regulatory sequences).  A regulatory element is a short segment of DNA involved in the regulation of transcription.  In bacteria, a short sequence within the mRNA, the ribosome-binding site provides a location for a ribosome to begin translation.  Each mRNA contains a series of codons (three nucleotides).  The first codon, which is very close to the ribosome binding site, is called the start codon.  The start codon is followed by many codons that form the sequence of amino acids within the synthesized polypeptide.  A stop codon signals the end of translation. Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Stages of Transcription  Transcription occurs in three stages: initiation; elongation; and termination.  These steps involve the interactions with RNA polymerase, the enzyme that synthesizes RNA molecules.  Initiation: The promoter functions as a recognition site for transcription factors.  The transcription factors enable RNA polymerase to bind to the promoter.  Following binding, the DNA is denatured to form an open complex.  Elongation: RNA polymerase slides along the DNA in an open complex to synthesize mRNA.  Termination: A terminator is reached that causes RNA polymerase and the primary transcript to dissociate from the DNA.  Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Stages of Transcription  The initiation stage in the transcription process is a recognition step.  The sequence of bases within the promoter is recognized by the transcription factors.  The transcription factors and RNA polymerase bind to the promoter when the DNA is in the form of a double helix.  For transcription to occur, the DNA strands must be separated into a bubble-like structure called the open transcription bubble.  This allows one of the two strands to be used as a template for the synthesis of a complementary strand of RNA.  When RNA polymerase reaches a terminator, the RNA polymerase and the newly made RNA transcript dissociate from the DNA.  Dr. Rami Elshazli  Transcription in Prokaryotes Associate Professor of Biochemistry and Molecular Genetics  Messenger RNA (mRNA) is transcribed from the base sequence within DNA and then directs the synthesis of polypeptides.  The type of DNA sequence known as a promoter gets its name due to it “promotes” gene expression.  Most of the promoter is located upstream from the site of transcription start site.  The bases in a promoter sequence are numbered in relation to the transcriptional start site.  This site is the first nucleotide used as a template for transcription and is denoted +1.  The bases preceding this site are numbered in a negative direction.  No base is numbered zero.  Most of the promoter is labelled with negative numbers.  Dr. Rami Elshazli  Conventional numbering Associate Professor of Biochemistry and Molecular Genetics system of promoters  The first nucleotide that acts as a template for transcription is designated +1.  The numbering of nucleotides to the left of this spot is in a negative direction, whereas the numbering to the right is in a positive direction.  The nucleotide that is immediately to the left of the +1 nucleotide is numbered −1, and the nucleotide to the right of the +1 nucleotide is numbered +2.  There is no zero nucleotide in this numbering system.  In many promoters found in E. coli, two sequences are located at approximately the −10 and −35 sites in the promoter, are important in promoting transcription.  The sequence at the −35 site is 5′–TTGACA–3′, and the one at the −10 site is 5′–TATAAT–3′.  The TATAAT sequence is called the Pribnow box after David Pribnow discovered it in 1975.  Dr. Rami Elshazli  Transcription in Prokaryotes Associate Professor of Biochemistry and Molecular Genetics  The most commonly occurring bases within a specific type of sequence is called the consensus sequences.  These sequences at (-35 and -10 sites) were recognized by proteins that initiate transcription.  The enzyme that catalyzes the synthesis of mRNA is called RNA polymerase.  In E. coli, the core enzyme is composed of five subunits.  The association of a sixth subunit, sigma (σ) factor with the core enzyme creates the RNA polymerase holoenzyme.  The holoenzyme is required to initiate transcription.  The primary role of σ factor is to recognize the promoter.  The σ factor Protein is the main transcription factor that influence the function of RNA polymerase.  Dr. Rami Elshazli  Initiation phase of transcription Associate Professor of Biochemistry and Molecular Genetics  After the six subunits assemble with each other, RNA polymerase holoenzyme binds loosely to the DNA and then slides along the DNA, much like a train rolls along tracks.  When the holoenzyme encounters a promoter, σ factor recognizes both the −35 and −10 sequences.  Hydrogen bonding occurs between nucleotides in the −35 and −10 sequences of the promoter and amino acid side chains in the helix-turn-helix motif of σ factor.  The process of transcription is initiated when σ factor within the holoenzyme binds to the promoter to form a closed complex.  For transcription to begin, the double-stranded DNA must be unwound to form an open complex.  This unwinding begins at the TATAAT sequence in the −10 site, which contains only A-T base pairs.  Dr. Rami Elshazli  Initiation phase of transcription Associate Professor of Biochemistry and Molecular Genetics  A-T base pairs form only two hydrogen bonds, whereas G-C pairs form three hydrogen bonds.  DNA in an AT rich region is more easily separated.  A short strand of RNA is made within the open complex, and then σ factor is dissociated from the core enzyme.  The σ-factor subunit of RNA polymerase holoenzyme recognizes the −35 and −10 sequences of the promoter.  The DNA unwinds at the −10 sequence to form an open complex, and a short RNA is made.  Then σ factor dissociates from the holoenzyme.  Dr. Rami Elshazli  Elongation phase of transcription Associate Professor of Biochemistry and Molecular Genetics  After the initiation stage of transcription is completed, the RNA transcript is made during the elongation stage.  During the synthesis of the RNA transcript, RNA polymerase moves along the DNA, causing it to unwind, creating an open complex as it moves.  The DNA strand known as the template strand is used to make a complementary copy of RNA.  As RNA polymerase moves along the DNA, it creates an open complex that is approximately 17 bp long.  The rate of RNA synthesis is about 43 nucleotides per second.  Behind the open complex, the DNA rewinds back into a double helix.  RNA polymerase moves along the template strand in a 3’ to 5’ direction, and RNA is synthesized in a 5’ to 3’ direction.  The complementarity rule is the same as the AT/GC rule except that U is substituted for T in the RNA.  Dr. Rami Elshazli  Elongation phase of transcription Associate Professor of Biochemistry and Molecular Genetics  RNA polymerase always connects nucleotides in the 5′ to 3′ direction.  RNA polymerase catalyzes the formation of a bond between the 5′-PO42− group on one nucleotide and the 3′−OH group on the previous nucleotide.  Genes A and B are transcribed from left to right, and the bottom DNA strand is used as a template.  Gene C is transcribed from right to left, and the top DNA strand is used as a template.  In all three cases, the template strand is read in the 3′ to 5′ direction, and the synthesis of the RNA transcript occurs in a 5′ to 3′ direction.  Dr. Rami Elshazli  Termination phase of transcription Associate Professor of Biochemistry and Molecular Genetics  Transcription is terminated by:  RNA binding protein.  Intrinsic terminator.  The end of RNA synthesis is referred to as termination.  The hydrogen bonding between the DNA and RNA within the open complex is preventing dissociation of RNA polymerase from the template strand.  Termination occurs when this short RNA-DNA hybrid region is forced to separate, thereby releasing RNA polymerase as well as the newly made RNA transcript.  In E. coli, two different mechanisms for termination have been identified.  The rho-dependent termination: an RNA-binding protein known as rho (ρ) protein is responsible for terminating transcription.  The rho-independent termination: other genes does not require the involvement of ρ protein for terminating transcription.  Dr. Rami Elshazli  Termination phase of transcription Associate Professor of Biochemistry and Molecular Genetics  The ρ-Dependent Termination.  A rho recognition site upstream from the terminator, called the rut site acts as a recognition site for the binding of rho (ρ) protein.  It functions as a helicase, a protein that can separate RNA-DNA hybrid regions.  After the rut site is synthesized in the RNA, ρ protein binds to the RNA and moves in the direction of RNA polymerase.  Then, the DNA codes an RNA sequence containing several G-C base pairs that form a stem-loop structure.  RNA synthesis terminates several nucleotides beyond this stem-loop.  A stem-loop, also called a hairpin, can form due to complementary sequences within the RNA.  Dr. Rami Elshazli  Termination phase of transcription Associate Professor of Biochemistry and Molecular Genetics  This stem-loop forms immediately after the RNA sequence is synthesized and quickly binds to RNA polymerase.  This binding results in a conformational change that causes RNA polymerase to pause in its synthesis of RNA.  This pause allows ρ protein to catch up to the stem-loop and pass through it to break the hydrogen bonds between the DNA and RNA within the open complex.  When this occurs, the completed RNA strand is separated from the DNA along with RNA polymerase.  Dr. Rami Elshazli  Termination phase of transcription Associate Professor of Biochemistry and Molecular Genetics  The ρ-Independent Termination: [Intrinsic termination]  The process of ρ-independent termination does not require ρ protein.  In this case, the terminator includes two adjacent nucleotide sequences.  One sequence promotes the formation of a stem-loop.  The second sequence, which is downstream from the stem loop, is uracil-rich sequence located at the 3′ end of the RNA.  The formation of the stem-loop causes RNA polymerase to pause in its synthesis of RNA.  This pausing is stabilized by the protein NusA that bind to RNA polymerase.  At the precise time that RNA polymerase pauses, the uracil-rich sequence in the RNA transcript is bound to the DNA template strand.  The binding of this uracil-rich sequence to the DNA template strand is relatively weak, causing the RNA transcript to spontaneously dissociate from the DNA and stop transcription.  Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics  Dr. Rami Elshazli  Transcription in Eukaryotes Associate Professor of Biochemistry and Molecular Genetics  Gene transcription in eukaryotes is more complex than it The Three RNA Polymerases in Eukaryotic Cells is in prokaryotes. Type of polymerase Genes transcribed  Eukaryotic cells are larger than bacterial cells and RNA polymerase I 5.8S, 18S, and 28S rRNA genes contain a variety of compartments known as organelles. RNA polymerase II mRNA genes snRNA, snoRNA, lncRNA, miRNA genes  The genetic material within the nucleus of eukaryotes is RNA polymerase III tRNA genes, and 5S rRNA genes transcribed by three RNA polymerase enzymes, snRNA, miRNA genes designated RNA polymerase I, II, and III. The rRNAs were named according to their “S” values, which refer to their rate of sedimentation in an ultracentrifuge. The larger the S value, the larger the rRNA.  Each of the three RNA polymerases transcribes different categories of genes.  RNA polymerase III: it  RNA polymerase I: it transcribes the genes for transcribes tRNA genes and 5S ribosomal RNAs (rRNAs) except for 5S rRNA. rRNA gene.  RNA polymerase II: it is responsible for the synthesis of  It also transcribes a few genes mRNAs (primary transcript). that produce other non-coding  It also transcribes the genes for snRNAs, which are RNAs, such as snRNAs and needed for RNA splicing. microRNAs.  It transcribes several genes that produce noncoding RNAs, including lncRNA, microRNAs, and snoRNAs.  Dr. Rami Elshazli  Transcription in Eukaryotes Associate Professor of Biochemistry and Molecular Genetics  DNA enters the enzyme through the jaw and lies on a surface within RNA polymerase termed the bridge.  The part of the enzyme called the clamp is thought to control the movement of the DNA through RNA polymerase.  A wall in the enzyme forces the RNA-DNA hybrid to make a right-angle turn.  This bend facilitates the ability of nucleotides to bind to the template strand.  Mg2+ is located at the catalytic site, which is precisely at the 3′ end of the growing RNA strand.  Nucleoside triphosphates (NTPs) enter the catalytic site via a  As RNA polymerase slides along the template strand, a pore. rudder, which is about 9 bp away from the 3′ end of the  The correct nucleotide binds to the template DNA and is RNA, forces the RNA-DNA hybrid apart. covalently attached to the 3′ end.  The DNA and the single-stranded RNA then exit under a small lid.  Dr. Rami Elshazli  Transcription in Eukaryotes Associate Professor of Biochemistry and Molecular Genetics  Protein-coding genes have two features: a core promoter and regulatory elements.  The core promoter is a short DNA sequence that is necessary for transcription to take place.  This core promoter typically consists of:  TATAAA sequence called the TATA box.  Enhancers are recognized by regulatory transcription  The transcriptional start site, where transcription begins. factors (RTFs).  Downstream promoter elements (DPEs).  RFTs are proteins that affect the ability of RNA polymerase to recognize the core promoter and begin  The TATA box is usually about 25 bp upstream from the the process of transcription. transcriptional start site, plays important role in determining the precise starting point for transcription.  RTFs fall into two broad categories: activators and repressors.  In eukaryotes, transcription is influenced by enhancers,  In bacteria, segments of DNA that are recognized by which are DNA segments, that contain one or more activators or repressors are called operators rather than regulatory elements. enhancers.  Dr. Rami Elshazli  Transcription in Eukaryotes Associate Professor of Biochemistry and Molecular Genetics  Activators are proteins that bind to genes at sites known as enhancers and stimulate the rate of transcription.  Repressors are proteins that bind to selected sets of genes at sites known as silencers and thus slow transcription.  DNA sequences such as the TATA box and enhancers exert their effects only on a particular gene.  Transcription of eukaryotic protein-coding genes is initiated when RNA polymerase II and general transcription factors bind to the core promoter.  Three categories of proteins are needed for basal transcription at the core promoter:  RNA polymerase II.  General transcription factors.  Complex called mediators.  RNA polymerase II: The enzyme that catalyzes the linkage of nucleotides in the 5′ to 3′ direction, using DNA as a template.  Dr. Rami Elshazli  General transcription factors Associate Professor of Biochemistry and Molecular Genetics Basal Transcription Factors Needed for initiation phase  Most eukaryotic RNA polymerase II enzymes are Name Number of subunits Major functions composed of 12 subunits. TFIID 12 - Recognizes TATA box near the transcription start  TFIID: it composed of TATA-binding protein (TBP) and point at the promoter site other additional subunits. TFIIB 1 - Positions RNA polymerase II at the start site of  It recognizes the TATA box of the core promoter of transcription TFIIA 2 - Stabilizes binding of TFIID eukaryotic protein-coding genes. TFIIF 3 - Stabilizes RNA polymerase interaction with TFIIB  Most eukaryotic RNA polymerase II enzymes are - Helps attract TFIIE and TFIIH composed of 12 subunits. TFIIE 2 - Attracts and regulates TFIIH  TFIID: it composed of TATA-binding protein (TBP) and TFIIH 10 - Unwinds DNA at the transcription start point. - Phosphorylates the RNA polymerase C-terminal other additional subunits. domain (CTD).  It recognizes the TATA box of the core promoter of - Releases RNA polymerase from the promoter. eukaryotic protein-coding genes. TFIID is composed of TBP and 11 additional subunits  TFIIA: it binds to TFIID and stabilizes its binding to the TATA box.  TFIIB: it binds to TFIID and then positions RNA polymerase II to bind to the core promoter.  Dr. Rami Elshazli  General transcription factors Associate Professor of Biochemistry and Molecular Genetics Basal Transcription Factors Needed for initiation phase  TFIIF: it binds to RNA polymerase II and stabilizes its Name Number of subunits Major functions ability to bind to TFIIB and the core promoter. TFIID 12 - Recognizes TATA box near the transcription start  It plays a role in the attraction of TFIIE and TFIIH to bind point at the promoter site to RNA polymerase II. TFIIB 1 - Positions RNA polymerase II at the start site of transcription  TFIIE: it plays a role in the formation and the TFIIA 2 - Stabilizes binding of TFIID maintenance of the open complex. TFIIF 3 - Stabilizes RNA polymerase interaction with TFIIB  It may exert its effects by facilitating the binding of - Helps attract TFIIE and TFIIH TFIIH to RNA polymerase II. TFIIE 2 - Attracts and regulates TFIIH TFIIH 10 - Unwinds DNA at the transcription start point.  TFIIH: it functions as a DNA translocase, tracking - Phosphorylates the RNA polymerase C-terminal domain (CTD). along DNA and leaving unwound DNA in its wake. - Releases RNA polymerase from the promoter.  It separates the DNA strands to convert the TFIID is composed of TBP and 11 additional subunits preinitiation complex to an open complex.  It phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II, which releases it from interacting with TFIIB, thereby allowing RNA polymerase II to release from the core promoter.  Dr. Rami Elshazli  General transcription factors Associate Professor of Biochemistry and Molecular Genetics  Mediator: A complex that mediates the effects of regulatory transcription factors on the function of RNA polymerase II.  The ability of mediator to affect the function of RNA polymerase II occurs via the carboxyl-terminal domain (CTD) of RNA polymerase II.  Mediator can influence the ability of TFIIH to phosphorylate CTD,  Complex mediator itself could phosphorylate CTD.  CTD phosphorylation is required to release RNA polymerase II from TFIIB.  Thus, mediator plays a key role in the ability of RNA polymerase II to switch from the initiation to the elongation stage of transcription.  Dr. Rami Elshazli  Transcription in Eukaryotes Associate Professor of Biochemistry and Molecular Genetics  Six different general transcription factors (GTFs) and a protein complex called mediator are needed for RNA polymerase II to initiate transcription of protein-coding genes.  These series of interactions leads to the formation of the open complex.  Transcription factor IID (TFIID) is a very large protein complex that first binds to the TATA box and DPEs and thereby plays a critical role in the recognition of the core promoter.  The binding of TFIIA to TFIID enhances TFIID’s ability to bind to the TATA box.  TFIID is composed of 12 subunits, including TATA-  After TFIID binds to the TATA binding protein (TBP), which box, it associates with TFIIB. directly binds to the TATA box,  TFIIB promotes the binding of and several other proteins RNA polymerase II and TFIIF. called TBP-associated factors (TAFs).  Dr. Rami Elshazli  Transcription in Eukaryotes Associate Professor of Biochemistry and Molecular Genetics  The binding of TFIIE, TFIIH, and mediator completes the assembly of proteins to form a closed complex.  In eukaryotes, the closed complex is more commonly known as the preinitiation complex.  TFIIH and mediator play a major role in the formation of the open complex.  TFIIH has several subunits that perform different functions:  It act as helicases, which break the hydrogen bonds between the two strands of the DNA and thus needed to form an open complex.  It hydrolyzes ATP and phosphorylates a domain in RNA polymerase II known as the carboxyl-terminal domain (CTD).  It releases the RNA polymerase II from the core promoter.  In addition, complex mediator could phosphorylate the CTD.  CTD phosphorylation breaks the contacts between TFIIB and RNA polymerase II.  TFIIB, TFIIE, TFIIH, and mediator dissociate, and RNA polymerase II is free to proceed to the elongation stage of transcription.  Dr. Rami Elshazli  Transcription in Eukaryotes Associate Professor of Biochemistry and Molecular Genetics  Eukaryotic mRNAs are modified by cleavage near their 3′ end with the attachment of a string of adenine nucleotides.  This process is called polyadenylation requires the transcription of a polyadenylation signal sequence that directs the cleavage of the mRNA.  Transcription via RNA polymerase II typically terminates about 500–2000 nucleotides downstream from the polyadenylation signal sequence.  After RNA polymerase II has transcribed the polyadenylation signal sequence, the RNA is cleaved just downstream from this sequence.  This cleavage occurs before transcriptional termination.  Two models have been proposed for transcriptional termination:  Allosteric model.  Torpedo model.  Dr. Rami Elshazli  Transcription in Eukaryotes Associate Professor of Biochemistry and Molecular Genetics  Allosteric model:  In this model, RNA polymerase II becomes destabilized after it has transcribed the polyadenylation signal sequence, and it eventually dissociates from the DNA.  This destabilization caused by the release of proteins that function as elongation factors or by the binding of proteins that function as termination factors.  Torpedo model:  In this model, it suggests that RNA polymerase II is physically removed from the DNA.  The region of RNA that is still being transcribed and is downstream from the polyadenylation signal sequence is cleaved by an exonuclease that degrades the transcript in the 5′ to 3′ direction.  When the exonuclease catches up to RNA polymerase II, this causes RNA polymerase II to dissociate from the DNA.  Dr. Rami Elshazli Associate Professor of Biochemistry and Molecular Genetics