Gene expression and regulation

1. Gene expression and regulation An overview

2. Central dogma of gene expression An organism may contain many types of somatic cells, each with distinct shape and function. However, they all have the same genome. The genes in a genome do not have any effect on cellular functions until they are "expressed". Different types of cells express different sets of genes, thereby exhibiting various shapes and functions.

4. Double helix structure of DNA Francis Harry Compton Crick James Dewey Watson The B form, the helix makes a turn every 3.4 nm, and the distance Between two neighboring base pairs is 0.34 nm. Hence, there are about 10 pairs per turn. 

5. A-DNA and Z-DNA I. A form DNA In a solution with higher salt concentrations or with alcohol added. The DNA structure may change to an A form, which is still right-handed, Every 2.3 nm makes a turn and there are 11 base pairs per turn. II. Z form DNA Another DNA structure is called the Z form, because its bases seem to zigzag. The DNA molecule with alternating G-C sequences in alcohol or high salt solution tends to have such structure.  Z DNA is left-handed. One turn spans 4.6 nm, comprising 12 base pairs. 

6. The morphology of DNA

7. Genome “Genome“ is the total genetic information of an organism. -For most organisms, it is the complete DNA sequence. -For RNA viruses, the genome is the complete RNA sequence, since their genetic information is encoded in RNA. What do you see through the microscope lens below? This is a real picture of a person's chromosomes - taken from a single cell, stained

8. What are chromosomes and why do we need them? • -Chromosomes are compact spools of DNA. • -over 3 feet (1 meter) long from end to end if stretch out • -enable all this DNA to fit in the nucleus of each cell. • Normally, we have 46 of these packages in each cell;

9. Chromatin -Chromatin is the substance which becomes visible chromosomes during cell division.    -Its basic unit is nucleosome, composed of 146 bp DNA and eight histone proteins. The structure of chromatin is dynamically changing, at least in part, depending on the need of transcription. -The 30 nm chromatin fiber is associated with scaffold proteins (notably topoisomerase II) to form loops. 

10. From chromatin to chromosome Each loop contains about 75 kb DNA.  Scaffold proteins are attached to DNA at specific regions called scaffold attachment regions (SARs), which are rich in adenine and thymine. The chromatin fiber and associated scaffold proteins coil into a helical structure which may be observed as a chromosome. •G bands are rich in A-T nucleotide pairs •R bands are rich in G-C nucleotide pairs.

11. Chromatin -In the metaphase of cell division, the chromatin is condensed into the visible chromosome.  -At other times, the chromatin is less condensed, with some regions in a "Beads-On-a-String" conformation. • In the less condensed state (low salt) • In the condensed state (linked by H1 and H5)

12. Overview of DNA Replication

13. DNA polymerases Three types of DNA polymerases exist in E. coli: DNA polymerase I, II and III.  A. The DNA polymerase I is used to fill the gap between DNA fragments of the lagging strand.  It is also the major enzyme for gap filling during DNA repair.  B. The DNA polymerase II is encoded by the PolB gene, which is involved in the SOS response to DNA damage. C. DNA replication is mainly carried out by the DNA polymerase III. 

14. DNA polymerase in Mammals There are five types of DNA polymerases in mammalian cells: , , , , and .  The  subunit is located in the mitochondria, responsible for the replication of mtDNA. Other subunits are located in the nucleus.  Their major roles : : synthesis of lagging strand. : DNA repair. : synthesis of leading strand. : DNA repair.

15. Telomerase -Bacteria do not have the end-replication problem, because its DNA is circular. In eukaryotes, the chromosome ends are called telomeres which have at least two functions: -to protect chromosomes from fusing with each other -to solve the end-replication problem. 

16. Model of Telomerase as an RNA-Reverse Transcriptase ComplexSee Lingner, J., Hughes, T. R., Shevchenko, A., Mann, M., Lundblad, V. and Cech, T. R. Reverse Transcriptase Motifs in the Catalytic Subunit of Telomerase. Science 276, 561-567 (1997)

18. Overview of Transcription Transcription is a process in which one DNA strand is used as template to synthesize a complementary RNA.

20. The entire transcription process • Binding of polymerases to the initiation site.  Eukaryotic polymerases have to rely on other proteins (transcription factors) Prokaryotic polymerases can recognize the promoter and bind to it directly, (ii) Unwinding (melting) of the DNA double helix. Prokaryotic polymerases have the helicase activity Eukaryotic polymerases do not. Unwinding of eukaryotic DNA is carried out by a specific transcription factor.

21. (iii) Synthesis of RNA based on the sequence of the DNA template strand. RNA polymerases use nucleoside triphosphates (NTPs) to construct a RNA strand. (iv) Termination of synthesis.  Prokaryotes and eukaryotes use different signals to terminate transcription.  [Note: the "stop" codon in the genetic code is a signal for the end of peptide synthesis, not the end of transcription.

22. Classes of RNA polymerases E. coli: An E. coli RNA polymerase is composed of five subunits: two  subunits, and one for each , ', and  subunit.  A. (151 kD) and ' (156 kD) are significantly larger than a (37 kD).  B. Several different forms of  subunits have been identified, with molecular weights ranging from 28 kD to 70 kD. The  subunit is also known as the  factor.  <Q> What’s the function of  factor?

23. Eukaryotes: There are three classes of eukaryotic RNA polymerases:  • I, II and III, each comprising two large subunits and 12- • 15 smaller subunits. B. The two large subunits are homologous to the E. coli  and ' subunits.  C. Two smaller subunits are similar to the E. coli subunit.  D. However, the eukaryotic RNA polymerase does not contain any subunit similar to the E. coli factor.  Therefore, in eukaryotes, transcriptional initiation should be mediated by other proteins.

24. RNA polymerases vs. DNA polymerase • Both RNA and DNA polymerases can add nucleotides to • an existing strand, extending its length.  However, there is a major difference between the two classes of enzymes:  RNA polymerases can initiate a new strand but DNA polymerases cannot. Therefore, during DNA replication, an oligonucleotide (called primer) should first be synthesized by a different enzyme.

25. RNA processing RNA processing is to generate a mature mRNA (for protein genes) or a functional tRNA or rRNA from the primary transcript.  Processing of  pre-mRNA involves the following steps: 1. Capping - adding 7-methylguanylate (m7G) to the 5' end. 2. Polyadenylation - adding a poly-A tail to the 3' end. 3. Splicing - removing introns and joining exons.

26. .

27. RNA Editing This enzyme changes a codon, CAA, in the middle of the original mRNA to the stop codon UAA, thereby causing early termination of the protein synthesis. 

29. Prokaryotes -Many prokaryotic mRNAs are polycistronic, namely, an mRNA encodes more than one peptide chain.  -The polycistronic mRNA should contain multiple initiating codons. -In the standard genetic code, the codon for both initiating and non-initiating methionine is AUG. To distinguish them, prokaryotes use a specific sequence located about 5-10 bases upstream of the initiation AUG.  The specific sequence, UAAGGAGG, is known as the Shine-Dalgamo sequence The 16S rRNA of the ribosome contains a sequence  which can pair with the Shine-Dalgamo sequence:  5'—UAAGGAGG (5-10 bases)AUG     mRNA 3'--AUUCCUCC........ 16S rRNA

30. Eukaryotes The mechanism used by eukaryotes to recognize the initiating AUG is not entirely clear. Nearly all eukaryotic mRNAs are monocistronic (encodes a single peptide). Eukaryotic ribosome may simply scan from the 5' cap and identify the first AUG as the initiation site.    However, some viral mRNAs are polycistronic or lack 5' cap.  Marilyn Kozak found that the following sequence may increase the effectiveness as an initiation site 5'--ACCAUGG-        mRNA

31. Protein production - In bacteria, there are 30-40 tRNAs with different anticodons.  • In animal and plant cells, about 50 different tRNAs are • found.  Problem - there are 61 codons coded for amino acids.  • Suppose each codon can pair with only a unique anticodon, • then 61 tRNAs would be needed.