1. Expression of the Viral Genome in Host Cells (How do viruses express their genomes?)

2. Review of Replication, Transcription, and Translation  These three processes are separated in time and space in eukaryotic hosts.  In prokaryotic hosts all three of these processes can occur simultaneously.  Replication  The DNA polymerases that elongate the chain have proofreading or editing functions.  Proofreading removes inappropriate nucleotide monophosphates at the 3’ end.

3. Replication  In double stranded linear DNA, 1 end of each strand has a free 5’ carbon and 1 end has a free 3’OH group.  The two strands are in the opposite orientation with respect to each other (antiparallel).  Adenines always basepair with thymines (2 hydrogen bonds) and guanines always basepair with cytosines (3 hydrogen bonds)

4. The Structure of DNA

6. Polymerase template and primer requirements  DNA polymerase cannot initiate synthesis on its own. It needs a primer to prime or start the reaction.

7. DNA synthesis  Synthesis can occur only in the 5’ to 3’ direction.

8. DNA synthesis  Remember that DNA replication is semiconservative:

9. Semiconservative DNA replication

10. Replication  Replication of the bacterial chromosome  Begins at the origin of replication and proceeds in both directions. Synthesis continues until the whole chromosome is copied.

11. Bacterial chromosome replication

12.  The rate of replication is controlled at the level of initiation. The polymerase doesn’t go faster to increase the replication rate.  Remember that DNA polymerase requires a primer to initiate synthesis and that it can only synthesize in the 5’ to 3’ direction.  Remember also that the two strands are antiparallel; one is in the 5’ to 3’ direction, while its complementary strand is in the 3’ to 5’ direction.  This results in one strand (using the 3’ to 5’ template) being synthesized continuously (leading strand) and the other strand (using the 5’ to 3’ template) being synthesized discontinuously (lagging strand).

13. Bacterial chromosome replication  Leading strand synthesis – an RNA polymerase called a primase synthesizes a small piece of complementary RNA 1-5 nucleotides long to prime the continuous 5’ to 3’ synthesis, by DNA polymerase III, of the leading strand. Note that the template is being exposed in the 3’ to 5’ direction as the replication fork opens up.

14. Leading strand synthesis

15. Bacterial chromosome replication  Lagging strand synthesis – As the replication fork opens up, the template for the lagging strand is being exposed in the 5’ to 3’ direction.  But synthesis cannot occur in the 3’ to 5’ direction.  Therefore, the lagging strand is made discontinuously in fragments of approximately 1000 nucleotides.  Each fragment is started by a primase which synthesizes a stretch of 1-5 nucleotides of RNA.  This serves as a primer for DNA polymerase III to extend.  The fragments that are made are called Okazaki fragments.  In both the leading and the lagging strands, the RNA made by the primase is eventually digested away by the 5’ to 3’ exonuclease function of DNA polymerase I.  DNA polymerase I then fills in, with DNA, the gap made by the RNA digestion and the fragments are joined by DNA ligase.

16. Lagging strand synthesis

18. Replication fork

20. Replication  Replication of eukaryotic DNA  Eukaryotic DNA is linear and much longer  In order to replicate rapidly, it must have many simultaneous replication forks. Therefore there is an origin of replication every 10-1000 um along the DNA.

21. DNA Replication  The requirement for a primer for DNA synthesis creates a problem for achieving complete replication of linear genomes  There is no mechanism to fill in the gap left when the RNA primer is removed at the 5’ end of the leading strand  When the replication fork reaches the other end, a similar problem arises with the lagging strand.  Further replication would result in smaller and smaller strands of DNA  Eukaryotic cells solve this problem through the use of telomeres at the ends of their linear chromosomes. They replicate outside the normal replication process, thus preserving the normal chromosome length.  How do viruses solve this problem?

22. End Replication Problem

23. Review of Replication, Transcription, and Translation  Transcription – In transcription, a strand of RNA is synthesized from a portion of the cell’s DNA which has opened up via the activity of an endonuclease.  Transcription requires RNA polymerase and a supply of RNA nucleotides.  RNA polymerase can synthesize RNA only in the 5’ to 3’ direction.  RNA polymerase does not have proof-reading function  RNA polymerase does not require a primer to initiate synthesis

24. Transcription  Only one strand of the DNA serves as a template for the RNA synthesis. This is called the sense strand and the other strand is called the anti-sense strand.  The region on the DNA where the RNA polymerase binds to begin transcription is called the promoter.  The region on the DNA where transcription ends is called the terminator and at this point RNA polymerase and the newly formed RNA fall off.  The DNA double helix then reforms.

25. Transcription

26. Transcription

27. Prokaryotic Transcription  Prokaryotic transcription – one RNA polymerase makes all three types of RNA (mRNA, tRNA, and rRNA)  Structural genes for metabolic pathways are physically contiguous and under the control of a single set of controlling elements (an operon).  The transcription product is polycistronic mRNA with no 5’ cap or 3’ poly A tail.  Transcription and translation are tightly coupled.

28. Simultaneous Transcription and Translation

29. Eukaryotic transcription  Eukaryotic transcription – each type of RNA is made using a different RNA polymerase  No operons; each gene is regulated by its own controlling elements  The transcription product is a monocistronic mRNA that has a 5’ 7-methylguanosine cap and a 3’ poly A tail, and that it is usually a spliced product of the initial RNA transcript.  Transcription and translation are separated in both time and space.  Translation requires identification of the initiation codon, AUG. A ribosome binding site must also be present. Termination of translation occurs at a nonsense or stop codon, UAG, UAA, or UGA.

30. Amplification steps in Viral Replication  DNA viruses – can either bring in their own DNA polymerase or they can use the host cell DNA polymerase for replication of the viral genome.  Similarly, they can bring in their own RNA polymerase for transcription or they can use the RNA polymerase of the host cell. In order for the virus to use the host cell machinery for replication and transcription, the viral genome must contain the appropriate controlling sequences. In order for the virus to use the host cell machinery for replication and transcription, the viral genome must contain the appropriate controlling sequences.

31. Amplification steps in Viral Replication  RNA viruses – Neither prokaryotic nor eukaryotic cells contain RNA dependent RNA polymerases, therefore, all RNA viruses must bring in their own enzymes for replication of their genomic RNA and for transcription.  Positive strand RNA viruses only need to bring in the gene that encodes the RNA dependent RNA polymerase (replicase) enzyme.  Negative strand RNA viruses must bring in both the gene that encodes the replicase enzyme and a copy of the enzyme (i.e. the protein) itself. WHY?

32. Expression of the viral genome  For both DNA and RNA viruses  Genes expressed before genomic replication are the early genes.  Genes expressed after genomic replication are the late genes.  The early gene products are usually involved in helping the virus to establish control of the host cell and in getting the viral genome ready for replication.  The late gene products are usually structural proteins or proteins involved in maturation and release of the virus.