This document summarizes key aspects of gene transcription including:
1. Transcription is important for regulating cellular function and aberrant control can cause disease.
2. In eukaryotes, transcription and translation are separated in space and time, and primary RNA transcripts undergo extensive processing.
3. Prokaryotic transcription involves RNA polymerase recognizing promoters and transcribing DNA into RNA with sigma factors providing specificity. Eukaryotic transcription involves three RNA polymerases and more complex promoters.
Transcription is the process in which a gene's DNA sequence is copied (transcribed) to make an RNA molecule.
RNA polymerase is the main transcription enzyme.
Transcription begins when RNA polymerase binds to a promoter sequence near the beginning of a gene (directly or through helper proteins).
RNA polymerase uses one of the DNA strands (the template strand) as a template to make a new, complementary RNA molecule.
Transcription ends in a process called termination. Termination depends on sequences in the RNA, which signal that the transcript is finished.
difference between Transcription in eukaryotes and prokaryotes kamilKhan63
In prokaryotes the transcription is simple while in eukaryotes the transcription is complicated or complex.
Occurrences
Prokaryotic transcription occurs in cytoplasm.
Eukaryotic transcription occurs in nucleus.
3. In prokaryotes mRNA is transcribed directly from the template DNA strand while in eukaryotes 1st pre-mRNA is formed and then processed to yield mature mRNA.
Transcription is the process in which a gene's DNA sequence is copied (transcribed) to make an RNA molecule.
RNA polymerase is the main transcription enzyme.
Transcription begins when RNA polymerase binds to a promoter sequence near the beginning of a gene (directly or through helper proteins).
RNA polymerase uses one of the DNA strands (the template strand) as a template to make a new, complementary RNA molecule.
Transcription ends in a process called termination. Termination depends on sequences in the RNA, which signal that the transcript is finished.
difference between Transcription in eukaryotes and prokaryotes kamilKhan63
In prokaryotes the transcription is simple while in eukaryotes the transcription is complicated or complex.
Occurrences
Prokaryotic transcription occurs in cytoplasm.
Eukaryotic transcription occurs in nucleus.
3. In prokaryotes mRNA is transcribed directly from the template DNA strand while in eukaryotes 1st pre-mRNA is formed and then processed to yield mature mRNA.
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1.Definition
2.Transcription is selective
3.Transcription in Prokaryotes
•Initiation
•Elongation
•RNA polymerase vs DNA polymerase
•Termination
4.Transcription in Eukaryotes
•Initiation
•Elongation
•Termination
•Post transcriptional modifications
For MBBS, BDS and General Biochemistry students, coding strand, sense strand, anti-sense strand, promoter, enhancers, silencers, TATA box, Goldberg Hogness box, alternative spilicing, post-transcriptional modification
Protein synthesis is the process whereby biological cells generate new proteins. Translation, the assembly of amino acids by ribosomes, is an essential part of the biosynthetic pathway, along with generation of messenger RNA (mRNA), aminoacylation of transfer RNA (tRNA), co-translational transport, and post-translational modification. Protein biosynthesis is strictly regulated at multiple steps. They are principally during transcription (phenomenon of RNA synthesis from DNA template) and translation (phenomenon of amino acid assembly from RNA). The cistron DNA is transcribed into the first of a series of RNA intermediates. The last version is used as a template in synthesis of a polypeptide chain. Protein will often be synthesized directly from genes by translating mRNA. A proprotein is an inactive protein containing one or more inhibitory peptides that can be activated when the inhibitory sequence is removed by proteolysis during post translational modification. A preprotein is a form that contains a signal sequence (an N-terminal signal peptide) that specifies its insertion into or through membranes, i.e., targets them for secretion. The signal peptide is cleaved off in the endoplasmic reticulum. Preproteins have both sequences (inhibitory and signal) still present. In protein synthesis, a succession of tRNA molecules charged with appropriate amino acids are brought together with an mRNA molecule and matched up by base-pairing through the anti-codons of the tRNA with successive codons of the mRNA. The amino acids are then linked together to extend the growing protein chain, and the tRNAs, no longer carrying amino acids, are released. This whole complex of processes is carried out by the ribosome, formed of two main chains of RNA, called ribosomal RNA (rRNA), and more than 50 different proteins. The ribosome latches onto the end of an mRNA molecule and moves along it, capturing loaded tRNA molecules and joining together their amino acids to form a new protein chain.
3. Transcription and translation in eukaryotic cells are separated in space and time. Extensive processing of primary RNA transcripts in eukaryotic cells.
4. Transcription of DNA into RNA by RNA polymerase---an overview 1. Requires DNA template, four ribonucleotide 5’ triphosphates, Mg +2 . 2. De novo synthesis: does not require a primer. Low fidelity compared to DNA polymerase: errors 1/10 4 -10 5 (10 5 higher than DNA polymerase). 3. Activity highly regulated in vivo : at initiation, elongation and termination. 4. The nucleotide at the 5’ end of an RNA strand retains all three of its phosphate groups; all subsequent nucleotides release pyrophosphate (PPi) when added to the chain and retain only their phosphate (red). 5. The released PPi is subsequently hydrolyzed by pyrophosphatase to Pi, driving the equilibrium of the overall reaction toward chain elongation. 6. Growth of the transcript always occurs in the 5’-to-3’ direction. Template (non -coding) strand Non-template (coding) strand
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7. E. coli RNA polymerase holoenzyme bound to DNA Subunit Stoichiometry Role in holoenzyme 2 Binds regulatory sequences/proteins Forms phosphodiester bonds/binds ribo- nucleoside triphosphate substrates ’ Promoter recognition RNAP assembly A single RNA polymerase makes multiple types of RNAs (rRNA, tRNA and mRNA) in prokaryotes. Known as Processive.
8. Typical E.coli promoters recognized by an RNA polymerase holoenzyme containing 70 Strong promoters: frequent initiation of transcription-every 2 seconds Weak promoters: may contain substitutions, transcribed every 10 min.
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10. DNase I footprinting: a common technique for identifying protein-binding sites in DNA. 1. A DNA fragment is labeled at one end with 32 P (red dot). 2. Portions of the sample then are digested with DNase I in the presence and absence of a protein that binds to a specific sequence in the fragment. 3. A low concentration of DNase I is used so that on average each DNA molecule is cleaved just once (vertical arrows). 4. The two samples of DNA then are separated from protein, denatured to separate the strands, and electrophoresed. The resulting gel is analyzed by autoradiography, which detects only labeled strands and reveals fragments extending from the labeled end to the site of cleavage by DNase I.
12. Dissociation of RNAP and purification of by ion-exchange chromatography Fraction number Carboxymethyl- (-CO 2 -2 ) or phospho- (-PO 3 -2 ) cellulose Absorbance at 280 nm ’ [NaCl] [protein] ’
13. The dissociable sigma subunit gives promoter specificity to prokaryotic RNA polymerase (RNAP) ’ Core enzyme + Holoenzyme No specific promoter binding; tight non-specific DNA binding (K d ~5 x 10 -12 M) Specific promoter binding; weak non-specific DNA binding (K d ~10 -7 M); finds promoter 10,000 times faster. ’
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16. Transcription initiation by prokaryotic RNA polymerase Core enzyme Holoenzyme Promoter -35 -10 “ sliding and scanning” Open complex; initiation Closed complex rNTPs PPi 5’pppA/G mRNA Sigma separates from the core once a few phosphodiester bonds are formed ’ ’ ’
17. Interactions of various sigma factors of E. coli with the same core polymerase to form holoenzymes with different promoter-binding specificity Sigma Factor Promoters Recognized Promoter Consensus -35 Region -10 Region 70 Most genes TTGACAT TATAAT 32 Genes induced by heat shock TCTCNCCCTTGAA CCCCATNTA 28 Genes for motility and chemotaxis CTAAA CCGATAT 38 Genes for stationary phase and stress response ? ? -24 Region -12 Region 54 Genes for nitrogen metabolism & other functions CTGGNA TTGCA Heat-shock response: High temperature induces the production of 32, which binds to the core polymerase to form a unique holoenzyme for recognition of the promoters of heat-shock induced genes.
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19. Transcriptional elongation: Movement of transcription bubble (17-bp, 1.6 turns of B-DNA duplex) Supercoiling of DNA during transcription causes a requirement for topoisomerases Speed of movement: 50-90-nt/sec
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21. Rho-independent prokaryotic transcription termination The core polymerase pauses after synthesizing a hairpin. If the hairpin is really a terminator, RNA will dissociate from the DNA strand as the A-U pairing is unstable. Once the RNA is gone, DNA duplex reforms and the core is driven off, as it has low affinity for dsDNA.
22. Rho-dependent transcription termination Platt , Ann. Rev. Biochem. 55 : 339 (1986) Rho-binding Site (non-contiguous structural features in RNA): Stop signals not recognized by RNAP alone. termination Rho: forms RNA-dependent hexameric ATPase, translocates along RNA 5’-to-3’, pulling RNA away when it reaches the transcription bubble.
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27. tRNA: cloverleaf; “L” model Attachment to mRNA here Stem region AA attaches here
37. First step in RNA processing: Capping Formation of the 7-methylguanine 5’- 5’ cap structure. The 5’ cap structure is essential for efficient pre-mRNA splicing, export, stability and translation initiation. Three separate enzyme activities are required for cap formation. Phosphatase Guanyl transferase Methyl transferase The cap protects the RNA from 5’-exonucleolytic cleavage *Note the unusual 5’ – 5’ linkage *Note the 7-methylguanine