Transcription Transcription- synthesis of RNA from only one strand of a double stranded DNA helix

1. Transcription Transcription- synthesis of RNA from only one strand of a double stranded DNA helix DNARNA(Protein) Why is RNA an intermediate???? Protect the DNA; limited access; Gives regulatory opportunity (all cells have the same DNA but not the same genes are expressed) 3. In Eukaryotes the DNA is located in the nucleus and RNA transports the information out to the protein synthesis apparatus in the cytoplasm

2. RNA transcription •  same general mechanism in prokaryotes and eukaryotes •  Differences occur in gene structure Prokaryotes  Genes usually code for more than one polypeptide (euks only one)  Introns almost non existent in prokaryotes consequently less mRNA processing in bacteria  Transcription and translation occur simultaneously in prokaryotes  all takes place in cytoplasm in prokaryotes

3. RNA synthesis is a template dependent process  The DNA dependent RNA polymerases adds ribonucleotide units to the 3' end of the growing RNA chain using one strand of the DNA duplex as a template  the added ribonucleotides adhere to the base pairing rules except for the addition of U instead of T  the RNA has a sequence identical to the non template strand of DNA except for substitutions of U for T  Polymerization occurs only in the 5' to 3' direction, as does DNA synthesis  No primer is required for transcription

4. The bacterial RNA polymerase  consists of a core enzyme containing several polypeptides  α, β, and β’ subunits make up the core. This core enzyme is potentially capable of copying DNA from any source to RNA  In order to initiate transcription with high efficiency on bacterial promoters, the enzyme must combine with another polypeptide unit, - a sigma factor  the sigma factor increases the selectivity of the bacterial RNA polymerase for bacterial promoters by ~ a million times  Distinct sigma factors enable RNA polymerases to distinguish between different bacterial gene groups - transcription regulation

5. RNA synthesis is initiated at specific DNA sequences called promoters  Promoters are part of bacterial transcription units called operons  Operons usually contain more than one coding sequence. An individual coding sequence in an operon is called a cistron  Most bacterial operons are controlled by a single promoter lying in the region in front of the transcribed fragment  Some have 2 or more promoters arranged tandemly in the 5' flanking region  different promoters are controlled by different regulatory factors

6. Consensus sequences • Bacterial promoters contain consensus sequences that provide recognition and binding sites for RNA polymerase, regulate the rate of initiation of transcription and indicate the start point  2 consensus sequences occur in E coli promoters that are recognised by RNA polymerase in combination with sigma factor σ70  the short sequence TATATT, also called the -10 sequence or Pribnow box, is usually centered about 10 nucleotides upstream of the start point.  TTGACA, called the -35 sequence, usually begins about 35 nucleotides upstream from the start point

7. Bacterial promoters  Vary in the strength with which they bind RNA polymerase  depends on how closely their -10 and -35 sequences fit the consensus sequence  those with perfect copies of the consensus sequence bind strongly  those with base substitutions altering the consensus sequence bind RNA polymerase more weakly  The degree of binding of RNA polymerase by the promoter sets the base level for the initiation of transcription  This base level is adjusted by 2 types of regulatory proteins called repressors and activators

8. Prokaryotic transcription proceeds through initiation, elongation and termination • Initiation  the core enzyme in combination with a sigma factor binds strongly to a promoter  strong binding is quickly followed by the DNA unwinding and the initiation of transcription at the start point • The sigma factor is released after transcription begins 2. Elongation • As elongation begins, NusA (a protein) is loaded on to the RNAP(RNA polym) complex and sigma factor is displaced from the complex. • the core enzyme continues transcribing the operon until it reaches termination signals at the 3' end of the gene

11. 3. Termination there are 2 types of sequence signalling termination • rho independent termination/intrinsic termination there is formation of a stable GC rich stem-loop in the newly synthesized RNA followed by a string of U's (A's in the template strand) spaced about 20 bases downstream (these sites are often called intrinsic terminators). The stem loop "snares" the polymerase, slowing or stalling it. This pause, coupled with the low stability of the RNA-DNA hybrid at the active site (run of A=U basepairs) allows the RNA polymerase to fall off the template DNA and terminates the RNA transcription for that gene. 2. rho dependent termination rho binds the newly formed RNA and stalls the RNA polymerase by interacting with it. Rho termination activity is stimulated by ATP hydrolysis. Nus factors (proteins) can interact to enhance termination

12. rho independent termination/intrinsic termination

13. rho dependent termination • A sequence near the end of the newly synthesized strand binds to the rho protein. This protein physically forces the release of the RNA.

14. The Types or Classes of RNA ·mRNA o      this is the coded information for how to make a particular protein §       codon = 3 nucleotide code specifying an amino acid o      monocistronic – codes for just one protein o      polycistronic – codes for several proteins o      mRNA code also has start and stop signals for translation o      may have an upstream leader sequence that also regulates activity o      unstable molecule ·  rRNA o      stable o      helps to make up the ribosomes o      bacteria have 16s, 23s, and 5s rRNA o      undergoes posttranscriptional modification (RNA processing): §       pieces are cut from a larger primary transcript ·  tRNA o      stable o      carries amino acids to the ribosome for protein synthesis o      has an anti-codon that base pairs with the mRNA codon o      must have a different tRNA for each amino acid o      RNA processing (see fig 8-9): §       Cut out tRNA from a much longer primary transcript §       Chemical modification of some bases