Transcription - Lecture Note

Transcription Overview of transcription Transcription produces a copy of the coding strand of DNA using ribonucleotides This process is known as transcription, because a copy of one strand of DNA, known as the coding strand, is transcribed into a single strand of ribonucleotides To produce a protein, the sequence of ribonucleotides in the RNA copy of a gene must then be read by the ribosome, which translates the information encoded by the bases into a polypeptide chain. The RNA is therefore named messenger RNA for its role in carrying a copy of the genetic information to the ribosome DNA is transcribed into RNA by an RNA polymerase enzyme, which makes a copy of the coding strand by using the complementary strand of DNA, known as the template strand The RNA polymerase enzyme catalyzes the formation of phosphodiester linkages, as successive ribonucleotides are added to the 3' end of the growing RNA chain Transcription proceeds through three key stages Transcription begins when RNA polymerase locates and binds to a DNA sequence immediately preceding the gene. This sequence is called the promoter and signals where transcription should start Since transcription proceeds in a 5' to 3' direction, sites located 3' to this are said to be located downstream, whereas those located to the 5' side are said to be upstream In the initiation phase, the RNA polymerase enzyme separates the two strands of DNA, creating a short unpaired region known as a transcription bubble RNA polymerase begins by synthesizing the first few ribonucleotides of the RNA transcript, while remaining at the promoter. In contrast with DNA polymerase, no primer is required to begin this process When RNA polymerase succeeds in synthesizing an RNA product of sufficient size, the enzyme moves past the promoter and changes conformation, becoming more stably associated with the DNA In this elongation phase, the enzyme unwinds the DNA ahead of it to expose the single-stranded template DNA. Behind the enzyme, the template strand pairs again with the coding strand to reform the DNA duplex Within this open region, a short stretch of the DNA template stand is base-paired with the most recently synthesized RNA ' while the rest of the growing RNA strand is extruded through RNA polymerase Elongation of the RNA transcript continues until RNA polymerase encounters a DNA sequence known as a terminator that signals where RNA synthesis should stop (the termination phase) The packaging of eukaryotic DNA into chromatin poses a barrier to transcription Recall that each nucleosome consists of 147 base pairs of DNA wrapped around a histone octamer. This wrapping can present a barrier to transcription because it can prevent RNA polymerase and other proteins from binding to DNA Eukaryotic cells overcome the chromatin barrier by using three general classes of enzymes that are also recruited to the transcribed gene, in addition to RNA polymerase (1) nucleosome-remodeling enzymes, which reposition histones and histone octamers from one location on the DNA to another, help remove the barrier to transcription imposed by the structure of chromatin and allow binding of the necessary proteins to the promoter (2) histone chaperones, which disassemble and reassemble the histone octamer, also help to remove the chromatin barrier from promoters and from the path of the elongating RNA polymerase enzyme (3) there are enzymes that catalyze reversible chemical modification of histone proteins. These modifications play a central role in modulating chromatin structure and in governing the recruitment of proteins that bind to particular histone modifications Acetylation, methylation, ubiquitination, sumoylation, phosphorylation, and adenosine diphosphate (ADP) ribosylation are examples of post-translational modifications found in histone proteins Transcription is regulated in all cells The amount of each protein and RNA molecule in the cell can be adjusted by controlling the number of RNA transcript made of each gene. Transcription is therefore A highly regulated process RNA Polymerase Core Enzyme RNA polymerase is a multisubunit enzyme with a conserved core Bacteria and archaea each possess a single type of RNA polymerase, while eukaryotes typically have three RNA polymerase enzymes that are each responsible for transcribing a different class of genes Functions of the eukaryotic RNA polymerase I directs transcription of large ribosomal RNA polymerases: RNA (rRNA) genes; RNA polymerase II directs transcription of all genes encoding messenger RNAs (mRNAs), and RNA RNA polymerase I —> rRNA polymerase III directs transcription of a variety of RNA RNA polymerase II —> molecules, including transfer RNA (tRNA) and 5S rRNA mRNA However, it is important to note that RNA polymerase I and RNA polymerase III —> III together are responsible for more than 80% of the total tRNA, 5S RNA, snRNA RNA in growing eukaryotic cells RNA polymerases IV, V At the heart of all RNA polymerase enzymes is a set of (plants only) —> siRNA subunits called the core enzyme, which catalyzes the synthesis of RNA using a DNA template Despite these variations, the RNA polymerase core enzymes from bacteria, archaea, and eukaryotes share the same basic architecture, reflecting a common mechanism for synthesizing RNA using a DNA template The bacterial core enzyme illustrates key features of RNA polymerases The bacterial core enzyme is a complex of roughly 400 kDa, comprising five polypeptides: two copies of the alpha subunit and one each of beta, beta prime, and omega In some bacteria, the large beta and beta prime subunits are produced as a single polypeptide. The alpha subunit is composed of an N-terminal domain and a C-terminal domain The C-terminal domain of the alpha subunit does not form part of the globular core enzyme, but rather extends outward where it is available for interactions with the DNA template and transcriptional regulators The active site of the RNA polymerase enzyme lies at the base of the long cleft separating the two jaws. The active site is also accessible to the outside of the jaw through an opening in the beta subunit Eukaryotic and archaeal RNA polymerases share a common central architecture with the bacterial enzyme All three eukaryotic RNA polymerases and the archaeal RNA polymerases contain a central core that is remarkably similar to that of bacterial RNA polymerase, even though the eukaryotic and archaeal enzymes are larger and contain more subunits RNA polymerase enzymes from all three domains of life contain five central subunits that are similar in amino acid sequence and three dimensional structure The additional subunits in RNA polymerase I and III may play roles that are carried out by general transcription factors in the case of RNA polymerase II The RNA polymerase II CTD couples transcription with pre-mRNA processing The RNA polymerase II enzyme, alone of the three eukaryotic polymerases, carries out an additional function - it couples transcription to processing of the RNA transcript to produce the mature mRNA The mRNA synthesized by RNA polymerase II is rarely in its mature, fully functional form. Instead, it is termed a pre-mRNA and most be subjected to various processing events that yield the final mRNA These chemical modifications include the addition of a guanosine cap to the 5' end of the nascent transcript, as well as the attachment of a polyadenosine (polyA) tail to the 3' end The coupling of transcription to RNA processing depends on an important feature unique to RNA polymerase II called the C-terminal domain Selected residues in the CTD are phosphorylated at different stages of the transcription cycle. The phosphorylated CTD then recruits RNA processing enzymes, which modify the RNA while it is being transcribed Promoter Recognition in Bacteria and Eukaryotes Sigma factors direct bacterial RNA polymerase to promoters Bacterial RNA polymerase core enzymes can transcribe RNA on their own, but they do not recognize promoter sequences. Instead, the bacterial core enzyme requires a specialized subunit called a sigma factor, which directly contacts the promoter sequence It is therefore the particular sigma factor utilized by a given RNA polymerase that determines which genes will be transcribed by that polymerase holoenzyme All bacterial cells have a primary sigma factor, which directs transcription from the promoters of genes encoding essential proteins that are needed by growing cells Each alternative sigma factor directs transcription of a set of genes that are only required under certain conditions or whose expression must be increased or decreased in response to changing conditions Bacterial species differ significantly in the number of sigma factors they encode, reflecting the different environmental conditions to which they must respond, and hence the different transcription programs that they must activate under particular conditions The large number of sigma factors enables these organisms to initiate transcription from different promoters at different times, depending on the specific sigma factors produced in the cell at a given moment Sigma factors bind to specific sequences that define bacterial promoters The sigma factor guides the bacterial RNA polymerase holo enzyme to to the promoter by binding to specific sequences upstream of the start site of transcription, whose position along the DNA sequence is denoted as +1 Bacterial promoters generally contain two key elements that are recognized by a sigma factor a conserved sequence most commonly centered at 35 nucleotides upstream of the start of transcription, and therefore termed the -35 (minus 35) element, and a second conserved sequence centered at ten nucleotides upstream of the start site known as the -10 (minus 10) element Variations in both promoter element spacing and sequence allow for differential regulation of genes, since these DNA sequence variations will have distinct effects on the relative binding of different classes of sigma factors Tighter binding to a particular promoter will lead to higher relative levels of transcription, which, in turn, will lead to higher levels of protein expressed, whereas weak binding to another promoter will result in lower rates of transcript production Some bacterial promoters contain sequences, in addition to the -35 to -10 regions, that can further modulate RNA polymerase binding and initiation of transcription At some promoters that lack an optimal -35 sequence, transcription initiation depends on sigma binding to an extended -10 region consisting of an additional two bases located on the upstream side of the - 10 region Some bacterial genes that are transcribed at a high rate contain an additional AT-rich sequence called the UP element located upstream side of the -35 region. The UP element serves as a binding site for the C-terminal domain of the alpha subunit of RNA polymerase When sigma binds to the RNA polymerase core enzyme to form the RNA polymerase holoenzyme, the three structural domains of sigma are positioned to recognize specific elements of the promoter sequence Recognizes the -35 region and is bound to a flexible flap of the polymerase beta subunit Binds to the - 10 region and promotes promoter welding Recognizes the two bases comprising the extended -10 region Sigma factor activity can be regulated in many ways While the amounts of the core subunits of RNA polymerase remain relatively constant under different growth conditions, the levels and activities of many sigma factors can vary significantly in response to environmental or developmental signals The relative amount of sigma factor in the cell can be regulated by modulating the transcription or translation of the sigma factor, or by modulating the stability of the sigma factor proem or of its mRNA transcript Some sigma factors are synthesized as pro-sigma factors, meaning that they contain inhibitory domains that must be cleaved off before the sigma factor can associate with the core RNA polymerase Eukaryotic and archaeal RNA polymerases require general transcription factors for promoter recognition These proteins are called general transcription factors and come together to form a large complex that binds RNA polymerase and helps it initiate transcription at specific promoters These factors, which are either single proteins or complexes, are named for the distinct, biochemically purified species that were required to reconstitute promoter - specific transition initiation in vitro However, just one protein is required for transcription initiation by all eukaryotic polymerases; this protein is called TATA binding protein (TBP), which is also a subunit of the TFIID complex Eukaryotic and archaeal promoters are often defined by a TATA box and other core promoter elements The promoters of eukaryotic genes contain a variety of sequences called core promoter elements that direct the binding of the transcription initiation complex, thereby determining the start site and direction of transcription Promoters are most commonly located in accessible regions of chromatin lacking nucleosomes, denoted nucleosome-free regions or NFRs, which leaves the DNA free for binding to the transcription initiation complex When present, the TATA box is centered approximately 25 - 30 bp to the upstream side of the transcription start site. A promoter element called BRE (for TFIIB recognition element) is the binding site for the transcription factor TFIIB TFIID nucleates assembly of the RNA polymerase II pre-initiation complex The first step in the assembly of the RNA polymerase II transcription initiation complex involves the binding to promoter DNA of general transcription factors, which position pol II for subsequent initiation For promoters with a TATA box, the initial step occurs when TFIID binds to the TATA box.TFIID plays the same role as the bacterial sigma factor in locating the promoter It [TBP] is a saddle-shaped protein that binds to the minor groove of DNA, with its concave face inducing profound bending and partial unwinding of the DNA double helix Once TFIID is bound to the promoter region, assembly of the pre-initiation complex proceeds with the recruitment of TFIIB, a single polypeptide that recognizes the adjacent BRE promoter element When TFIIB binds to the RNA polymerase core enzyme, an extended linker, called the B reader that connects the N-terminal and C-terminal domains of TFIIB, extends deep into the active site of the enzyme Assembly of the pre-initiation complex proceeds with the recruitment of the core RNA polymerase II enzyme, which is in a complex with TFIIF. This is the followed by the binding of TFIIE and TFIIH, thus yielding the complete transcription pre-initiation complex Additional protein complexes are required for transcription by RNA polymerase II Additional protein complexes, however, are required for transcription of most genes in vivo. These include a variety of enzymes that act on chromatin, as well as a complex known as mediator Mediator interacts with transcriptional activator proteins that are bound to upstream regulatory regions, promoting assembly of the transcription initiation complex at the promoter Changes in chromatin structure therefore are needed for transcription initiation, thus clearing the way for the assembly of the pre-initiation complex and allowing RNA polymerase to transcribe through chromatin RNA polymerases I and III use TBP to recognize their characteristic promoters RNA polymerases I and III, however, recognize quite distinct promoter sequences and have unique general transcription factors In each case, TBP is a subunit of a larger complex - SL1, TFIID, and TFIIB, complexes that are required for transcription by RNA polymerases I, II, and III, respectively Initiation of Transcription and Transition to an Elongating Complex RNA polymerase must separate the two strands of DNA RNA polymerase opens up a short region of around 14 bases known as the transcription bubble. The unpaired template strand binds to the polymerase near the active site and is kept apart from the non-template strand by regions of the RNA polymerase called the "rudder", "lid", and "zipper" The complex of RNA polymerase that is bound to an opened region of DNA and poised to initiate transcription is called the open complex Eukaryotic RNA polymerase II depends on the helicase subunits of the general transcription factor TFIIH, to unwind and separate the two DNA strands in a process that is fueled by hydrolysis of ATP The requirement of ATP-derived energy to separate the DNA strands is unique to RNA polymerase II; it is not required by RNA polymerases I or III or by the archaeal enzyme, for whom the change from closed to open complex occurs spontaneously RNA polymerase initiates synthesis of the nascent RNA chain The 3' OH of the last base in the growing RNA chain initiates A nucleophilic attack on the innermost phosphate of the incoming ribonucleoside triphosphate. This is an energetically favorable reaction that reads to formation of a phosphodiester bond with the 5' carbon of the newly added nucleotide and release of pyrophosphate The 5' end of the growing RNA chain exits through A special exit channel, while a loop within the lid helps to separate the RNA from the DNA template Just like the DNA polymerase, RNA polymerase has two Mg 2+ ions in the active site that play key roles in activating the 3' OH for nucleophilic attack and stabilizing a developing negative charge on the leaving oxygen Once the new phosphodiester bond is formed, pyrophosphate is released and then further broken down by hydrolysis, releasing additional energy and thereby favoring the polymerization reaction Surprisingly often, RNA polymerase fails to synthesize the full-length RNA on the first attempt and instead enters into a series of abortive cycles of synthesis. These cycles, referred to as abortive initiation, result in the production and release of short RNA products, which are subsequently degraded Displacement of the loop (or B finger) is thought to help the polymerase enzyme break away from the promoter by disrupting contacts with proteins that helped to recruit the core polymerase, setting the stage for the next step in the transcription cycle - promoter clearance RNA polymerase undergoes a conformational change as it leaves the promoter During this process, termed, promoter clearance, the polymerase enzyme undergoes a conformational change that closes the clamp region around the DNA and helps makes the association of the enzyme with the DNA very stable At the same time, RNA polymerase loosens its grip on initiation factors - the bacterial sigma subunit or the eukaryotic general transcription factors - leading to their eventual release Eukaryotic RNA polymerase II becomes phosphorylated, as it converts from the initiating complex to the elongating complex. Specifically, the general transcription factor TFIIH phosphorylates the CTD of the large subunit Rpb1, at the serine that is the fifth residue of the heptad repeat Transcription Elongation Transcription proceeds in a highly protective manner Once RNA polymerase has created the promoter region and made the transition to an elongating complex, transcription proceeds in a processive manner, such that one nucleotide after another is added in quick succession by a single RNA polymerase enzyme for hundreds Or even thousands of bases Favorable interactions between RNA polymerase and the RNA-DNA hybrid formed by the recently transcribed RNA and the DNA template strand further stabilize the transcribing polymerase enzyme on the DNA Elongation factors suppress the pausing of transcription and rescue stalled polymerase enzymes Instead, the polymerase pauses periodically during RNA synthesis, most often due to physical obstructions that prevent the smooth passage of the polymerase along the DNA strand One type of sequence-dependent pausing occurs when the RNA transcript contains short complementary stretches that form a transient hairpin Transcriptional pausing can be brought about by the presence of a weak DNA - RNA hybrid in the transcription bubble, the result of an AU-rich sequence or a misincorporated base The polymerase can eventually resume RNA synthesis on its own, although pausing can either he relieved or enhanced by proteins called elongation factors Pausing is promoted by the negative elongation factors NELF and DSIF, which are thought to stabilize the paused polymerase on the DNA template Pausing and arrest of transcription can be alleviated by cleavage of the RNA strand This [backtracking] causes the most recently synthesized portion of the RNA to separate from the template strand and protrude through the funnel region of RNA polymerase as the transcription bobble moves backward Transcription can be restarted with the assistance of transcript cleavage factors, which assist RNA polymerase in cleaving of the protruding 3' end of the RNA transcript and allow transcription to resume Transcript cleavage has been postulated to have a role in error correction; if mismatched bases are incorporated into the growing mRNA, they may destabilize the RNA - DNA hybrid and produce distortions in the duplex region, which causes the RNA polymerase to stall Transcript cleavage factors promote the hydrolysis of the phosphodiester bond, thus releasing the unpaired RNA nucleotides at the 3’ end of the transcript Nucleosomes must be removed from the path of the elongating polymerase for transcription to proceed Just as nucleosomes can interfere with the assembly of the transcription pre-initiation complex on DNA, they also pose a barrier to the elongating RNA polymerase enzyme The DNA must be unwrapped from the histone core in order to open a transcription bubble, yet this has an energetic cost that could cause the elongating polymerase to stop transcribing This [removing nucleosomes] is accomplished with the help of histone chaperones, which aid I, the temporary disassembly and reassembly of histone octamers The need to dismantle and then reassemble nucleosides during transcription has been established primarily for RNA polymerase II Transcription elongation produces supercoiling that must be relieved by topoisomerases As the transcription bubble moves through a double-stranded DNA template, RNA polymerase is continually unwinding the DNA template at the downstream end of the enzyme. At the same time, the unpaired strands at the upstream end must twist around one another to re-form the DNA double helix Unwinding the downstream DNA therefore reduces the twist of the DNA, which is compensated for by an increase in positive supercoiling ahead of the transcribing polymerase Similarly, the reannealing DNA behind the polymerase enzyme increases in twist as the double helix re-forms, leading to an accumulation of negative supercoils in the upstream regions The build-up of superhelical tension could cause the elongating complex to stall and therefore must be relieved. This is achieved through the action of topoisomerase enzymes Transcription Termination Transcription is terminated by a variety of mechanisms Once RNA polymerase has succeeded in transcribing a complete gene or set of genes, the enzyme must stop transcribing at the proper position and release both the completed transcript and the DNA This prevents inappropriate read-through transcription of genes further downstream and frees the polymerase to initiate transcription again In essence, the effect of terminators on transcription is opposite to that of promoters - a promoter is a defined sequence that promotes transcription, whereas a terminator is a defined sequence that stops it Intrinsic terminators promote termination in the absence of additional factors At certain terminator sites, RNA polymerase terminates transcription in the absence of any other factors. These DNA sites are called intrinsic or simple terminators One is [bacterial intrinsic terminator] an inverted repeat sequence that specifies a secondary structural element in the transcribed RNA in which the RNA strand folds back on itself and basepairs to form a hairpin-like structure called a stem loop The second feature is a series of around 8-10 adenosine residues (A) (polyadenosine or poly (A)) immediately downstream that encodes a stretch of uridine residues (U) (poly(U)) in the RNA Once RNA polymerase has transcribed the terminator sequence, the complementary halves of the inverted repeat in the RNA transcript base-pair with each other and form the stem-loop structure The opening up of this region [poly (U)-poly (A) RNA-DNA hybrid] within the transcription bubble is thought to arrest transcription, while the RNA hairpin part of the terminator sequence interacts with the polymerase and somehow helps to pull the RNA out of the active site, causing the elongation complex to dissociate Termination of some transcripts requires additional protein factors At these sites, denoted enzymatic terminators, additional protein factors are required for RNA polymerase to terminate transcription and be released from the DNA template The terminators of rho-dependent genes do not contain the characteristic hairpin and run of U residues that is found in intrinsic terminators Rho is a hexameric ATPase that binds to C-rich sites on the nascent RNA chain termed rut sites (for rho utilization). The rho hexamer, can exist as an open ring, which facilitates its loading onto the RNA The activity of rho also helps to displace the RNA from the DHA and prevent the formation of RNA -DNA hybrids known as R-loops, in which several base pairs of the RNA hybridize with the template strand, thus displacing the complementary strand of DNA Polyadenylation of mRNA is linked to transcription termination As the nascent transcript exits the polymerase enzyme, the mRNA is cleared at the poly (A) signal and the 3' end of the mRNA is processed, yielding a polyadenylated transcript According to the "allosteric" model, recognition of the poly (A) or 3'- end processing signals, along with RNA cleavage at the processing sites, could read to a conformational change in the polymerase enzyme that causes it to dissociate from the DNA template and release the remaining 3' RNA fragment