Ms. Shivani Bhagwat Lecturer, School of Biotechnology DAVV Transcription of DNA

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  • Ms. Shivani Bhagwat Lecturer, School of Biotechnology DAVV Transcription of DNA
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  • Transcription: information transfer from DNA to RNA The information encoded by DNA is transcribed into RNA information of RNA is translated into proteins Transcription (RNS synthesis) is carried out by RNA polymerases RNA polymerases are large complexes of proteins, not single enzymes Transcription is more complex in eukaryotes than in prokaryotes, but basic mechanisms are very similar in all living cells Protein coding sequences constitute only 1-2 % of the human genome! DNA encodes a number of RNA molecules, which are not mRNAs: ribosomal RNAs, rRNAs transferRNAs, tRNAs small nuclear RNAs, snRNAs small nucleolar RNA, snoRNA microRNAs, miRNAs anti-sense RNAs RNA molecules, that are parts of enzymes
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  • Information transfer from DNA to RNA Transcription and translation are closely linked processes. In prokaryotes they are physically linked, in eukaryotes they mutually regulate each other, sharing controling molecules and mechanisms. Cells contain 20 times more RNA than DNA
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  • Transcription, RNA polymerases DNA is transcribed by RNA polymerase, a large protein complex, which synthesizes an RNA strand, complementary to the template strand of DNA. RNA polymerase has to unwind the DNA over a short distance then moves stepwise along the template strand of DNA. The newly formed RNA is only basepaired with a short stretch of deoxynucleotides. RNA polymerase enzymes are very large: 100 kDa in T7 bacteriophage. 400 kDa in bacteria. 500 kDa for eukaryotes
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  • RNA polymerases RNA polymerase adds nucleotides one by one to the RNA chain at the polymerisation site. The polymerase rewinds the two DNA strands behind this site to displace the newly formed RNA. A short region of DNA/RNA helix is therefore formed only transiently and the RNA transcript is a single- stranded complementary copy of one of the two DNA strands. The incoming nucleotides are in the form of ribo- nucleoside triphosphates (ATP, UTP, CTP, and GTP), whose hydrolysis provides the energy for the polymerisation reaction.
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  • RNA polymerases All multi-subunit RNA polymerases have 5 core subunits. The (true) Bacteria have an additional -(sigma) factor subunit that aids regulation and binding to DNA. Eukaryotic RNA polymerases have five core subunits plus five common subunits. Plastid and mitochondrial RNA polymerases and bacteriophage T7's RNA polymerase are single-subunit DNA polymerases
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  • The prokaryotic holoenzyme has 2 alpha, 2 beta and an omega subunits. It binds DNA with no sequence specificity. To find the promoter sequence it needs another subunit: the sigma factor Formation of the open complex from closed DNA is usually the slowest step in transcription initiation. About 10 nucleotides are polymerized in the open complex before it must undergo promoter clearance. A conformational change must take place that releases the sigma factor. RNA polymerases
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  • Prokaryotic promoter sequences Promoter -35 Region spacer -10 Region start trp operon TTGACA N17 TTAACT N7A tRNA tyr TTACA N16 TATGAT N7A lP2 TTGACA N17 GATACT N6G lac operon TTTACA N17 TATGTT N6A rec A TTGATA N16 TATAAT N7A lex A TTCCAA N17 TATACT N6A T7A3 TTGACA N17 TACGAT N7A consensus TTGACA TATAAT TGAC box (-35) TATAAT Box (-10), This sequence was initially called the Pribnow Box. Do not refer this sequence as a TATA box. All these promoter sequences are recognized by the sigma70 subunit in E. coli. Note the degree of sequence variation at each position. The consensus sequence has been derived from a much larger database of over 300 well-characterized promoters. The "consensus sequence" is a hypothetical sequence made up of the nucleotides found most often in each position. There may be no single organism with this exact set of nucleotides in the stated positions. The promoter sequences are asymmetrical, the promoter will bind the polymerase in only one orientation, thus determining which strand of the DNA will be transcribed.
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  • RNA synthesis by RNA pol RNA polymerase synthesizes ribosomal RNAs. In bacteria there are many (in E. coli 7) ribosomal rRNA genes. RNA polymerase as all other NA synthesizing enzymes polymerizes from 5>3, DNA is read from 3>5 The produced RNA is complementary to template strand, and equivalent to non-template strand RNA polymerase stops at termination or stop signal. Rho factor binds RNA and helps recognizing termination signals (RNA loops). Palindromic sequences (e.g. AATCG.CGATT) form loops on the RNA and serve as signal termination.
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  • The 16S rRNA The 16S rRNA is 1.5 kb long and has an elaborate secondary structure
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  • rRNA molecules in the ribosome Ribosomes are very large RNA-protein complexes, subcellular organelles. They are responsible for the synthesis of proteins. Both prokaryotic and eukaryotic ribosomes consist of two subunits and have 3 (4) RNA molecules.
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  • Processing of the rRNAs Endonucleolytic cleavage of ribosomal RNA precursors in E. coli. The primary transcript contains a copy of each of the three ribosomal RNAs and may also contain several tRNA precursors. The large rRNA precursors are cleaved from the large primary transcript by the action of RNase III. The ends of the 16S, 23S, and 5S rRNAs are trimmed by the action of endonucleases M16, M23, and M5, respectively. (Slash marks indicate that portions of the rRNA primary transcript have been deleted for clarity.)
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  • Processing of tRNA molecules tRNAs and other small RNA molecules are produced by processing and modification of larger precursor molecules. This is the processing of a bacterial tRNA.
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  • Synthesis of prokaryotic mRNAs Genes coding for proteins are first transcribed into mRNAs. One gene codes for a protein. In cases of multi-subunit proteins with heterolo- gous subunits, one cistron codes for a polypeptide chain. (A protein with four different subunits is coded by four cistrons.) In prokaryotes there are single cistronic and polycistronic messages, coding for one protein or a chain of (related) enzymes. Metabolically linked enzymes (e.g. enzymes of a synthetic pathway) are usually controlled together. Polycistronic mRNAs are characteristic of prokaryotic organisms.
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  • Prokaryotic regulation of transcription There are genes, which are expressed constitutively. Others are under control: synthetic enzymes are negatively regulated (switched off) if their product is present, enzymes with catabolic functions are induced (swithed on) if their substrate is available. Bacteria with continuously changing environment have more regulatory units, than those, which live in more stable niche.
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  • The lac operon
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  • Regulatory elements of the eukaryotic promoter A TATA Box, located approximately 25 bp upstream of the startpoint of transcription is found in many promoters. The consenus sequence of this element is TATAAAA, it resembles the TATAAT sequence of the prokaryotic -10 region (please do not mix them up!) TATA box appears to be more important for selecting the startpoint of transcription (i.e. positioning the enzyme) than for defining the promoter. The Initiator is a sequence that is found in many promoters and defines the startpoint of transcription. The GC box is a common element in eukaryotic class II promoters. Its consensus sequence is GGGCGG. It may be present in one or more copies which can be located between 40 and 100 bp upstream of the startpoint of transcription. The transcription factor Sp1 binds to the GC box. The CAAT box - consensus sequence CCAAT - is also often found between 40 and 100 bp upstream of the startpoint of transcription. The transcription factor CTF or NF1 binds to the CAAT box.
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  • The eukaryotic RNA pol I In eukaryotes RNA polymerase I transcribes rRNA genes. The CORE promoter region is located from -31 to +6 around the transcription startpoint. Other sequences further upstream, called the up- stream control elements (UCE) in the promoter- proximal region located from -187 to -107 are also required for efficient transcription.
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  • The eukaryotic RNA pol I Assembly of a eukaryotic transcriptional complex. Two additional transcription factors are known to be required to assist RNA polymerase I. UBF1 is a single polypeptide which binds to the upstream control element (UCE) and to the CORE promoter. UBF1 recognizes a GC-rich sequence within these elements. UBF1 is an assembly factor. SL1 binds to UBF1. It consists of 4 proteins, one of which is TATA-box binding protein (TBP). TBP is required for the assembly of a transcriptional complex in all 3 classes of eukaryotic transcription units. SL1 is a positional factor - it targets RNA polymerase at the promoter so that it initiates transcription in the correct place. Once UBF1 and SL1 have formed a complex, RNAP I binds to the CORE promoter to initiate transcription
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  • RNA polymerase II RNA polymerase II All genes that are transcribed and expressed via mRNA are transcribed by RNA polymerase II. Transcription copies the DNA code of a gene and converts it to high mol mass nuclear RNA (hnRNA), which is precessed to mRNA. The mRNA will be used at the ribosome to make polypeptides (proteins). RNA polymerase II is a multisubunit enzyme-comp