Transcription BIOLOVE ed. 1


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Transcription processes

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Transcription BIOLOVE ed. 1

  1. 1. TRANSCRIPTION RNA polymerase carries out transcription. Transcription is the process of linking ribonucleotide to form long chain or to form oligonucleotide. The process of synthesizing RNA from DNA molecule. The 2’-OH of the ribose sugar make phosphodiester bond unstable and that causes ssRNA easy to be degraded. Cell has mechanism to remove mRNA that is not correct. Transcription VS Replication - Transcription initiates both of prokaryotic and eukaryotic cell Form more sites than replication The number of RNAP molecules are more than number of DNAP molecules RNAP proceed at slower rate than DNAP (~ 50-100 bases/sec for RNAP, ~ 1000 bases/sec for DNAP) The fidelity of RNAP lower than DNAP  Allowed since the aberrant RNA molecules can simply be turned over and new correct molecules can be made. (true for Prok. But not Euk.) **Replication happens too fast and cause lots of error but DNAP has exonuclease activity to correct the error. Transcription has three steps (Initiation, Elongation and Termination) Before proceeds to the steps, let us look at the Promoter. Promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located near the genes they transcribe, on the same strand and upstream on the DNA (towards the 3' region of the anti-sense strand, also called template strand and non-coding strand). Promoters can be about 100–1000 base pairs long. Promoters contain specific DNA sequences and response elements that provide a secure initial binding site for RNA polymerase and for proteins called transcription factors that recruit RNA polymerase. In bacteria - The promoter is recognized by RNA polymerase and an associated sigma factor, which in turn are often brought to the promoter DNA by an activator protein's binding to its own DNA binding site nearby. In eukaryotes - The process is more complicated, and at least seven different factors are necessary for the binding of an RNA polymerase II to the promoter. Nik_mal 2013 Page 1
  2. 2. TRANSCRIPTION In bacteria, the promoter contains two short sequence elements approximately -10 and -35 nucleotides upstream from the transcription start site.   The sequence at -10 (the -10 element) has the consensus sequence TATAAT. The sequence at -35 (the -35 element) has the consensus sequence TTGACA. The nucleotide at which transcription is initiated is denoted as +1. The preceding nucleotide as -1. Those portions of the DNA preceding the initiation site (toward the 3’ end of the template) are said to be upstreamfrom that site. Those portions of the DNA succeeding it (toward the 5’ end of the template) are said to be downstream from that site. The RNAP is a holoenzyme that consist of 5 subunits Rifampicin – a semi synthetic antibiotic derived from the original molecule (rifamycin – isolated from streptomycete). Binds to the beta (β) subunit of RNAP and inhibits the formation of first phosphodiester bond. Act on prok. and euk. The sigma (σ) factor - Proteins that are required for the binding of the RNA polymerase to the promoter so the transcription can be initiated. Recognizes promoter and facilitates initiation. Facilitate the binding of RNAP to dsDNA. To direct RNAP to transcribe DNA template at the correct transcription start site (TSS). Function of sigma (σ) factor - Ensure RNAP binds stably to DNA only at promoter (not string bind) at first but then if reach prmoter sequence the strong binding) Destabilize nonspecific binding to nonpromoter DNA Stabilize specific binding to promoter DNA Accelerates the search for promoter DNA Nik_mal 2013 Page 2
  3. 3. TRANSCRIPTION RNA polymerases are not capable of recognizing promoters on their own but require the help of additional proteins called transcription factors (in Eukaryotes) The polymerase moves along the template DNA strand towards its 5’ end (3’  5’ direction). Therefore, the resultant pre-mRNA transcript grows from the 5’ end. The DNA is temporarily unwound as the polymerase progresses and reform the double helix after the polymerase has moved past a particular stretch of DNA. Chain elongation occurs as a result of an attack by 3’ OH of the nucleotide at the end of the growing strand on the 5’ α-phosphate of the incoming nucleoside triphospahte For each gene, only one of the two DNA strand is transcribed. This strand is called the template strand. Note that for every particular gene, the same strand is used as the template every time it is transcribed. The mRNA base triplets are called codons. the nontemplate strand is sometimes called the coding strand. The nontemplate sequences are same as the sequence of mRNA except for the T is replaced by U. Nik_mal 2013 Page 3
  4. 4. TRANSCRIPTION Transcription in Prokaryotes As what has been mentioned before, bacteria such as E.coli contain RNA polymerase composed of 5 subunits. Without the σ subunit, it is called as core enzyme. Before the enzyme (RNAP) attaches itself to the DNA, σ factor is added. Attachment of σ factor to the core enzyme increases the enzyme’s affinity for promoter sites in DNA and decreases its affinity for DNA in general. Once bound to the promoter, the enzyme separates (melt) the two DNA strands in the region surrounding the start site, which make the template strand accessible to the enzyme’s active site. One approximately ten nucleotides have been successfully incorporated into a growing transcript; the enzyme undergoes a conformation change which lead to the release of σ sigma factor and the promoter DNA. This will make the enzyme more stable and is converted into an elongation complex. Bacterial cells possess a variety of different σ factors that Recognize different versions of the promoter sequence. The σ70 is known as the “housekeeping” σ factor, because it initiates transcription of most genes. Alternative σ factors initiate transcription of a small number of specific genes that participate in a common response. For example, when E. coli cells are subjected to a sudden rise in temperature, a new σ factor is synthesized that recognizes a different promoter sequence and leads to the coordinated transcription of a battery of heat-shock genes. To terminate the transcription, a specific nucleotide sequence is needed. Terminations involve two cases, which are rho-dependent termination and rho-independent termination (also known as intrinsic termination). Rho (ρ)-independent termination These termination sequences have two characteristics features: - A series of U residues in the transcribed RNA GC rich self-complementary region with several intervening nucleotides When the self-complementary region of a growing RNA chain is synthesized, the complementary sequences base-pair with one another, forming a stem-loop structure (hairpin loop). Nik_mal 2013 Page 4
  5. 5. TRANSCRIPTION This stem-loop structure is thought to interact with the transcribing E.coli RNA polymerase, causing it to pause. The base pair between the U residues at the 3’ end of the nascent RNA chain and the A residues in the template DNA strand are extremely unstable compared to other types of WatsonCrick base pairs. If the base pairs is replaced or substituted with other bases, the terminator function may decreases because the RNA-DNA hybrid duplex will be more stable. These two features probably permit release of the RNA chain from the transcription complex at Rho-independent termination sites. Rho (ρ)-dependent termination - - Rho factor is a hexameric protein which wraps around the 70-to 80-base segment of the growing RNA transcript Activates the ATPase activity of Rho that is associated with its movement along the RNA in the 3’ direction until it eventually unwinds the RNA-DNA hybrid (break H-bond) at the active site of RNAP. Pausing of polymerase during elongation is thought to be an important component of Rhodependent termination as it is in Rho-independent termination. Nik_mal 2013 Page 5