Learning Objectives for Gene Regulation Lecture.
by Dr. Ilya Ioshikhes,
Department of Biomedical Informatics, 3017 Graves Hall,
Tel. 292-6514, E-mail: ioschikhes-1@medctr.osu.edu
Eukaryotes and prokaryotes.
1.
2.
3.
4.
5.
6.
What is the meaning of these terms?
Name some typical organisms that belong either to prokaryotes and eukaryotes.
What is the main difference between the prokaryotes and eukaryotes?
What is a cell hereditary substance?
How is it organized in prokaryotes and eukaryotes?
What is a chromosome? How many chromosomes are in Human? In E. coli?
DNA, RNA and proteins – basics.
7. What are the building blocks of DNA?
8. What nucleotides are involved in DNA and RNA structure?
9. How they are bound?
10. What is a difference between the DNA and RNA?
11. Name major functions of the DNA and RNA.
12. How the information is transferred from DNA to proteins?
13. What are the building blocks of proteins?
14. How they are bound?
15. What is the primary factor determining a protein’s shape and structure?
16. Do the DNA, RNA and protein molecules have a beginning and an end? What are
they?
17. What is the major DNA structure?
18. What are the rules of complementarity for the DNA?
From DNA to proteins.
19. What is the central dogma of molecular biology?
20. What are genes?
21. What are the main steps of gene expression in prokaryotes?
22. – “ – in eukaryotes?
23. What is the difference?
Promoters and transcription.
24. What is the DNA transcription?
25. What is the promoter of a gene?
26. What are its major elements in prokaryotes?
27. –“– in eukaryotes?
28. What is the TATA box and where it is located?
29. What other core promoter elements do you know?
30. What is RNA Polymerase and what are its functions?
31. What types of the RNA Polymerase do you know? Which type participates in
mRNA synthesis?
32. What are general transcription factors and their function?
33. What are cis- and trans-acting promoter elements? Examples?
34. What are possible roles of transcription factors (TF)?
35. Describe common bioinformatics methods for finding of TF binding sites.
36. What are enhancers and how they act?
Nucleosomes and chromatin.
37. What is the chromatin?
38. What are the nucleosomes?
39. What is their primary role?
40. Which basic levels of chromatin organization do you know?
41. What is the role of nucleosomes in gene regulation?
42. How the transcription is initiated if most of the promoters covered by the
nucleosomes?
DNA methylation and CpG islands.
43. What is the DNA methylation?
44. How it may transform the DNA sequence?
45. How it may influence the gene expression?
46. What are the CpG islands and where they are located?
Splicing.
47. What are exons and introns?
48. What is primary RNA transcript?
49. What is RNA splicing?
50. What is alternative splicing?
51. What are possible roles of the alternative splicing in a gene expression?
Translation.
52. What is the translation in molecular biology?
53. Describe its major components.
54. What is the role of RNA in protein synthesis?
55. Which forms of RNA participate in it?
56. Bring an example of a translational regulation.
Key information elements.
Eukaryotes and prokaryotes.
1. Cells are the basic structural units of most of the living matter.
2. Most of the organisms (excepting viruses) are a cell (single-celled organisms) or a
complex of multiple cells (multi-cellular organisms).
3. The two major cell types are: eukaryotic cells (literally those with a true nucleus)
and prokaryotic cells (those lacking a defined nucleus). All multi-cellular
organisms are eukaryotes.
4. Eukaryotic cells contain a number of organelles.
5. Prokaryotes and eukaryotes contain similar macromolecules. DNA is a hereditary
substance of the both types.
6. The organization of DNA differs greatly in prokaryotic and eukaryotic cells. In all
prokaryotes studied to date, most or all of the cellular DNA is in the form of a
single circular molecule. The nuclear DNA of all eukaryotic cells, in contrast, is
divided between two or more different chromosomes. Human somatic cells
contain two sets of 23 chromosomes.
7. Each chromosome contains a single linear double-stranded DNA molecule, bound
to various proteins.
DNA, RNA and proteins – basics.
8. Nucleic acids (DNA and RNA) are linear polymers of nucleotides connected by
phosphodiester bonds.
9. A nucleotide has three parts: a phosphate group, a pentose (a five-carbon sugar
molecule), and an organic base.
10. In RNA, the pentose is always ribose, in DNA – deoxyribose.
11. The name of the bases (and often of nucleotides) are adenine, guanine, cytosine
(found in both DNA and RNA); thymine (found only in DNA); and uracil (found
only in RNA) – abbreviated as A, G, C, T, and U, respectively.
12. The bases are of two kinds: purines (A and G, their structure is two fused rings)
and pyrimidines (C, T and U, with only one ring).
13. In oligonucleotides, the nucleotide at one end has a free 3’ (deoxy)ribose
hydroxyl group, at the other end – a free 5’ phosphate or hydroxyl. They are
related to the 3’- and 5’-ends of the molecule, respectively.
14. The native state of DNA is a double helix of two antiparallel chains that have
complementary sequences of nucleotides. The bases on the opposite strands are
held together by hydrogen bonds: A is paired with T (2 bonds), G is paired with C
(3 bonds).
15. The building blocks of proteins are 20 amino acids. Each contains an amino group
(-NH2) (imino group –NH- in proline), carboxyl group (-COOH) and a side chain
(R group). The amino acids differ only in their side chains.
16. Polypeptides are polymers composed of amino acids connected by peptide bonds.
Proteins are polypeptides or complexes thereof.
From DNA to proteins.
17. Transcription of DNA to RNA to protein: This central dogma forms the backbone
of molecular biology and is represented by four major stages. 1. The DNA
replicates its information in a process that involves many enzymes: replication.
2. The DNA codes for the production of messenger RNA (mRNA) during
transcription. 3. In eukaryotic cells, the mRNA is processed (essentially by
splicing) and migrates from the nucleus to the cytoplasm. 4. The messenger RNA
carries coded information to ribosomes. The ribosomes "read" this information
and use it for protein synthesis. This process is called translation. Proteins do not
code for the production of protein, RNA or DNA. They are involved in almost all
biological activities, structural or enzymatic.
18. Gene is a DNA segment responsible for a synthesis of certain protein. It is a basic
unit of heredity. It has specific position in DNA molecule (certain chromosome in
eukaryotes). Complex of all genes of certain organism is its genome.
19. Prokaryotic mRNAs are frequently polycistronic: They encode multiple proteins,
each of which is translated from an independent start site. Eukaryotic mRNAs are
usually monocistronic, encoding only a single protein.
20. There are DNA segments not involved in any gene – non-coding DNA. It is
frequently called “junk” DNA, but often has important regulatory functions. The
non-coding DNA occupies majority of the human genome.
21. Many mechanisms of the gene regulation are facilitated by “junk” DNA.
Promoters and transcription.
22. The copying of DNA into RNA is called transcription.
23. DNA has two strands, but usually only one of them participates in the
transcription, from 5’- to 3’-end of the DNA.
24. Specific DNA sites, at which RNA synthesis can begin, called promoters. They
are positioned upstream of the genes.
25. The transcription is initiated at a particular DNA sequence point called
Transcription Start Site (TSS). Some eukaryotic promoters have singular TSS,
others – multiple or alternative TSS-s.
26. In prokaryotes, there are clusters of genes controlled at one promoter site –
operons.
27. The major sequence elements in bacterial promoters are Pribnow (TATAAT) box
positioned ~ -10bp upstream of TSS and TTGACA motif at ~ -35bp.
28. In eukaryotic promoters, the most important sequence elements are TATA-box at
~ -30bp and Initiator (Inr) situated on TSS.
29. There are multiple other promoter sequence motifs (cis-elements) which serve as
substrates for trans-acting transcription factors (TF). None of them (including
TATA box and Inr) is really a universal element.
30. All transcription of DNA in bacteria is catalyzed by single RNA polymerase.
Eukaryotic cells have three different polymerases that make different classes of
RNA in the cell nucleus. The synthesis of mRNA is made by RNA polymerase II.
31. RNA polymerase II acts in cooperation with general transcription factors.
32. Other TFs have a regulatory role, including activation and repression of the
transcription.
33. Enhancers stimulate transcription. They may be positioned further upstream of the
promoters.
34. All promoter sequence elements are degenerated (not perfect). Common
bioinformatics methods for their description are by consensus sequence or by
positional weight matrix (PWM).
Nucleosomes and chromatin.
35. Chromatin (and chromosomes) is complex of DNA and proteins. Its general
structure is similar in all eukaryotic cells.
36. The basic unit of chromatin structure is nucleosome – complex of 8 protein
histone molecules (4 different kinds, 2 copies each) in the center (histone core)
and ~145bp of DNA wrapped around.
37. The length of the eukaryotic DNA is much larger than the size of the nucleus. In
order to fit the nucleus, DNA must be effectively packed. Nucleosomes constitute
the basic level of the packing.
38. Higher levels of chromatin organization include nucleosome arrays, chromatin
fibers and loops, and ultimately the chromosomes.
39. Being positioned along the promoters, nucleosomes have also a regulatory
function.
40. Histone modifications (acetylation and methylation) lead to chromatin activation
and remodeling and facilitate promoter activity.
DNA methylation and CpG islands.
41. Cytosine residues in vertebrate DNA can be modified by the addition of methyl
groups at the 5-carbon position.
42. DNA is methylated specifically in the CpG dinucleotides.
43. The DNA methylation often leads to the mutation C -> T in the CpG
dinucleotides (which are then transformed to TpG). The vertebrate genome as a
whole is therefore CpG-depleted.
44. Some DNA segments have preserved C and CpG content in the levels closed to
those statistically expected. If long enough, they are called CpG islands. CpG
islands are often positioned in the promoter area.
45. Thus methylation is correlated with reduced transcriptional activity of genes that
contain high frequencies of CpG dinucleotides in the vicinity of their promoters.
Splicing.
46. Many of the eukaryotic genes contain two or more protein-coding exons and
intervening non-coding introns.
47. In prokaryotic cells, translation of an mRNA into protein can begin from the 5’
end of the mRNA even while the 3’ end is still being copied from DNA.
48. In eukaryotic cells, the primary RNA transcript of a protein-coding gene must
undergo several modifications, collectively termed RNA processing, that yield a
functional mRNA. This mRNA then must be transported to the cytoplasm before
it can be translated into protein.
49. The final step in the processing of many different eukaryotic mRNA molecules is
splicing: the internal cleavage of the RNA transcript to excise the introns,
followed by ligation of the coding exons.
50. The expression of some proteins is regulated by controlling the processing of the
primary transcript from the gene encoding them. This type of gene regulation is
especially common for genes encoding proteins important for the function of the
nervous systems of vertebrates.
51. Alternative splicing of the primary transcripts produced from complex
transcription units often is regulated. As a result, different mRNAs may be
expressed from the same gene in different cell types or at different developmental
stages.
Translation.
52. The messenger RNA (mRNA) is translated into protein by the joint action of
transfer RNA (tRNA) and the ribosome, which is composed of numerous proteins
and two major ribosomal RNA (rRNA) molecules.