DNA Transcription

1. DNA Transcription Chapter 12

2. DNA Transcription: • The Central Dogma of Genetics • Promoters, Transcription Factor, and RNA Polymerases • Steps of Transcription • Post-Transcriptional Modifications, of RNA

3. Central Dogma of Molecular Biology • The flow of information in the cell starts at DNA, which replicates to form more DNA. Information is then ‘transcribed” into RNA, and then it is “translated” into protein. The proteins do most of the work in the cell. • Information does not flow in the other direction. This is a molecular version of the incorrectness of “inheritance of acquired characteristics”. Changes in proteins do not affect the DNA in a systematic manner (although they can cause random changes in DNA.)

4. Central dogma of molecular biology *1 *3 *2 *1 Reverse transcriptase allows RNA to be used to make DNA e.g. HIV replicates its RNA genome to DNA once in host cell *2RNA can also be used to make copies of RNA e.g. replication of viral RNA genome *3 Hypothetical

5. Genetic’s Central Dogma Central Dogma of molecular genetics: • DNA codes for RNA = transcription • RNA codes for protein = translation • DNA is the informational molecule which specifies RNA intermediate • RNA transmits the genetic information to protein with a genetic code in translation

6. Genetic Information Flow (Central Dogma)

7. Transcription- DNA directed RNA synthesis Transcription is the mechanism by which a template strand of DNA is utilized by specific RNA polymerases to generate one of the three different types of RNA (mRNA, tRNA and rRNA) What is the biological significance? • Allows selective expression of genes • Regulation of transcription controls time, place and level of protein expression

8. The 3 main types of RNA messenger RNA (mRNA): • Serve as the intermediate form of genetic information that is translated into polypeptide. • At any given time, they make up a small percentage of total cellular RNA, but they are the most diverse group (largest # of different species). • In the coding region ("open reading frame") of the mRNA, each amino acid is encoded by a triplet of bases (a codon).

9. The 3 main types of RNA transfer RNA (tRNA) • These small RNAs serve as the link between the codon and the amino acid it encodes. • One end (anticodon loop) is complementary to the codon and can pair with it. • The other end (acceptor end) forms is linked to an amino acid via an ester linkage to the -carboxylic acid.

10. The 3 main types of RNA ribosomal RNA (rRNA): • These form large RNA-protein complexes called ribosomes that are intimately involved in the translation process. • They serve as the surface on which the mRNAs are paired with tRNAs, and the amino acid are joined by peptide bonds.

11. "Other RNAs" • In fact, there are other RNA species in the cell besides these. • The ribosome is the most abundant (and largest and most complex) example of an RNA-protein complex. • There are other RNA-protein complexes involved in diverse processes in the cell. • Each one has a unique and specific RNA that forms an essential part of the RNA-protein complex's structure (e.g. telomerase).

12. Opening of the DNA double helix is required to form a transient DNA-RNA hybrid duplex as polymerization of the RNA chain proceeds. • Ribonucleoside triphosphates (NTP) are required as substrates. • They will pair with one of the DNA strands in Watson-Crick fashion: A-U and G-C base pairs.

13. Anatomy of a gene A typical protein-encoding gene can be divided into the following elements: • Transcriptional control sequences:bind transcription factors that can activate or inhibit transcription • Promoter: recruits RNA polymerase and marks the start of the transcribed region • Transcript: corresponds to the RNA sequenceCan be further subdivided into: • 5' untranslated region (5'UTR): before open reading frame – often has sequences that control translation • open reading frame (ORF): sequence translated into polypeptide • 3' untranslated region (3'UTR): after ORF; sometime has sequences that control translation • Termination sequences: required to terminate transcription

14. Prokaryotic transcription (E. coli) • RNA polymerase holoenzyme (') : complex stability (bind UP sequences) : catalytic subunit ': DNA binding (electrostatic) : binds promoters • The RNA pol holoenzyme is made by association of RNA pol core (') with a  subunit. Holoenzyme is able to recognize promoters.

15. Prokaryotic transcription (E. coli) • RNA pol is thought to scan the chromosome by binding nonspecifically to DNA; the  subunit provides specificity. • Certain DNA-binding proteins can also increase the frequency/efficiency with which promoters are used. • There is a consensus sequence for a promoter recognized by the standard  subunit (70): -35 region: TTGACA -10 region: TATAAT

16. Basal transcription set by promoter sequences: Note that no promoter actually has the consensus -10/-35 sequences. Promoter strength is correlated with similarity to the consensus sequences – the more the divergence, the weaker it is. This sets the promoter's level of "basal transcription" – in the absence of other factors.

17. Promoters Regions on DNA where RNA polymerase binds to initiate transcription.

18. Prokaryotic transcriptional regulation Transcription factor (TF): Any protein that initiates or regulates transcription In Prokaryotes: promoters are recognised by the basal transcription apparatus • This is the complex of TFs that assemble to recruit RNA polymerase • A protein called σ factor is required for promoter binding • The exchange of different σ factors and the re-targeting of RNA pol to a different promoter is the basis of selective gene expression in prokaryotes • Basal apparatus + RNA pol holoenzyme = Inititation complex

19. Initiation • RNA polymerase binds to promoter & opens helix B. Polymerisation De novo synthesis using rNTPs as substrate Chain elongation in 5’-3’ direction C. Terminationstops at termination signal

20. Stages in the process Initiation: Closed promoter complex RNA pol binds the promoter. Open promoter complex The DNA is unwound (about 1-1.5 turn of the helix from the -10 region to +2/+3) to allow one strand to act as template. Initiation 2 NTPs are joined together: N1TP + N2TP –> pppN1-p-N2 + PPi • This stage is often thought to be complete after polymerization of 8-9 nucleotides. This is called promoter clearance: the  subunit dissociates and RNA pol leaves the promoter. • Elongation commences

22. Stages in the process Elongation: • The core polymerase continues using the DNA template strand as a template and adds to the growing RNA strand. • Behind the polymerization site is an RNA-DNA duplex that is only ~1 turn of helix (8-9bp); the other DNA strand will reform a DNA duplex with the template strand after that, displacing the RNA. • A total of ~17 nt of the coding strand is displaced by the "transcription bubble." • The transcription bubble will be preceded by positive supercoils and followed by negative supercoils. Both are relieved by topoisomerases. • RNA pol moves at a rate of ~50-90 nt/sec.

23. Stages in the process Termination: • RNA pol is removed from the gene (otherwise the entire genome would be transcribed). • This usually requires a secondary structure in the sequence of the 3'UTR (most often a stem-loop) and • Rho termination factor (-dependent) • a poly-U sequence (-independent)