1. Genetics
FROM DNA TO PROTEINS

2. How does information get from DNA to Proteins?
Facts:
DNA resides in the nucleus
However, proteins are made in the cytoplasm
In the mid 1950’s, Paul Zamecnik determines that proteins were made at the ribosome.
But DNA …………………. Ribosome? How?

3. Discovery of RNA polymerase
1960 – Hurwitz and Weiss discovered RNA polymerase
RNA polymerase from E. coli is composed of five proteins (big molecule)
In eukaryotes, three RNA polymerases:
RNA polymerase I – makes rRNA
RNA polymerase II – mRNA
RNA polymerase III – small RNA molecules tRNA and one rRNA called 5S RNA

4. How does RNA Polymerase carry out its function?
1. Search the DNA template for promoters (sites on the DNA where the polymerase binds to start transcription.) Interact with other proteins that regulate transcription Unwind a short stretch of the DNA to expose single stranded DNA to copy into RNA Select the correct RNA nucleotides, based on the DNA sequence, and assemble the RNA chain Recognize termination signals and stop synthesizing RNA when a termination signal is detected.

5. The Central Dogma DNA ------------->RNA------------->Protein
The various cell types in a multicellular organisms owe their distinctive phenotypes to differences in the proteins made in each kind of cell.

7. Remember all DNA is not used to make proteins.
…”it is an astonishing fact that protein-coding sequences, which are what we mostly mean when we say “genes,” occupy only a little over 1% of the human genome.”
Some is used to make RNA; some functions as promoters, terminators, enhancers, etc.

8. What about proteins? Made of amino acids
Each amino acid has an amino group (NH2) and an acid (carboxyl) group COOH attached to a central H and a “R” group which distinguishes each amino acid.
There are 20 naturally occurring aa’s.

9. R groups impart particular functions in bonding and shaping proteins.
Some are polar, nonpolar, and ionized (acidic and basic).
Proteins have three levels of structure:
Primary – chain of amino acids lined by peptide bonds (determined by DNA)
Secondary – beta sheets and alpha helices (H-bonds)
Tertiary – folding by interactions between R groups
Quarternary – two or more chains interacting

10. Determining polarity of amino acids:
Remember, the polarity of
the R groups determines the
folding of the protein in the
tertiary level.

13. The shape of the protein determines its function.

14. What is the code?
Three nucleotides code for an amino acid – the nucleotide triplet of mRNA is called a codon.
Genetic code charts give the amino acids.
Methionine and tryptophan have a single codon.
Nearly all proteins begin with methionine.
Three codons code for “stop”.

16. The redundancy of the code
Since most amino acids have more than one codon, single-base changes in DNA often do not change the amino acid in the protein.

17. So how are proteins made?
Transcription – DNA to mRNA
Translation – Amino acids assembled to make proteins

18. DNA TRANSCRIPTION mRNA (a) Bacterial cell Fig. 17-3a-1
Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information
(a) Bacterial cell

19. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide
Fig. 17-3a-2
DNA
TRANSCRIPTION
mRNA
Ribosome
TRANSLATION
Polypeptide
Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information
(a) Bacterial cell

20. Differences in Eukaryotic and Prokaryotic Protein Synthesis
1. In a eukaryotic cell, the nuclear envelope separates transcription from translation
2. Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA. A primary transcript is the initial RNA transcript from any gene.

21. TRANSCRIPTION
Transcription copies short stretches of the coding regions of DNA to make RNA.
Different genes may be copied into RNA at different times in the cell's life cycle.
This process is catalyzed by the enzyme RNA Polymerase.
The basic transcription process is more or less similar in prokaryotes and eukaryotes, though the regulation of transcription is much more elaborate in eukaryotes.    

22. How does RNA Polymerase carry out its function?
1. Search the DNA template for promoters (sites on the DNA where the polymerase binds to start transcription.) Interact with other proteins that regulate transcription Unwind a short stretch of the DNA to expose single stranded DNA to copy into RNA Select the correct RNA nucleotides, based on the DNA sequence, and assemble the RNA chain Recognize termination signals and stop synthesizing RNA when a termination signal is detected.

23. How is RNA Polymerase like DNA Polymerase?
Like DNA polymerase, RNA Polymerase synthesizes new strands only in the 5' to 3' direction.

24. How is RNA Polymerase different from DNA Polymerase?
RNA Polymerase doesn't require a primer to start making RNA.
RNA Polymerase uses ribonucleotides, not deoxyribonucleotides.

25. How does an RNA polymerase know where to start copying DNA to make a transcript?
Signals in DNA indicate to RNA polymerase where it should start and end transcription. These signals are special sequences in DNA (consensus sequences) that are recognized by the RNA polymerase or by proteins that help RNA polymerase determine where it should bind the DNA to start transcription.
A DNA sequence at which the RNA polymerase binds to start transcription is called a promoter.

26. Eukaryotic polymerases are much more dependent that prokaryotes on additional accessory factors, called transcription factors, for everything from binding to the promoter to initialization of the polymerization reaction.

27. What does a promoter look like?
Bacterial promoters are probably the easiest to define. They are located close to the start of transcription (called the +1 site). There are two relatively conserved DNA sequences, located in two patches upstream (toward the 3' end of the template).
The first patch is located about 35 bases upstream of the +1 site, while a second often less conserved site is located about 10 base upstream.
One of the subunits of the bacterial polymerase binds to the -35 sequence.

29. Consensus sequences
Although most promoters vary in sequence, short stretches of common nucleotides called consensus sequences exist. They possess considerable similarity or consensus.
The existence of consensus in a set of nucleotides usually implies that the sequence is associated with an important function.
Ex – TATAAT, TTGACA both are common in bacterial promoters

30. Watch Nicolay explain how to decipher consensus sequences.

32. This shows the relative percentage of those nucleotides that are conserved in this consensus TATA box.

33. This is a consensus sequence for a transcription factor.

34. These regions of the promoter are important not only for determining where transcription starts,
but they also influence how often and how fast a gene is transcribed.

35. How does RNA polymerase bind and carry out transcription in prokaryotes?
Prokaryotic RNA polymerases have 2 components, a core enzyme and a sigma factor.
The sigma factor is necessary for the RNA polymerase to bind tightly to the promoter and initiate transcription.
Once transcription starts, the sigma factor falls off, and the core enzyme continues copying the DNA into RNA till it reaches a terminator. A terminator is a sequence of DNA that signals RNA polymerase to stop transcribing.

37. What about eukaryotic transcription?
In eukaryotes, the DNA template exists as chromatin, not as free DNA. The packaging of the DNA must therefore be "opened up" to allow access for transcription.
We have already considered how chromatin remodeling complexes and histone modifications can make DNA regions accessible for transcription.

38. What do eukaryotic promoters look like?
Eukaryotic promoters differ depending on which type of polymerase binds them. For RNA polymerase II, an important sequence located about 25 bases upstream is called the TATA box, because of the abundance of thymine and adenine bases in the sequence.
In general though, the promoters of eukaryotic genes are much more loosely defined, and therefore more difficult to recognize.

39. However eukaryotic promoters are alike in that they are bound by specific transcription factors that also influence how often the gene is transcribed.
Upstream (and downstream) regulatory sequences—sequences that a protein binds to and regulates the transcription of the gene—are also important for regulating eukaryotic transcription. These sequences can be far away.

40. TFIIA, TFIIB and so on are transcription factors.
They help guide RNA polymerase to the site.

41. This is a transcription unit.

42. Differences in Prokaryotic and Eukaryotic Transcription
Prokaryotes
Promoters close to transcription site
Uses a sigma factor to start transcription until termination segment is reached
Eukaryotes
DNA has to be opened up
Type of promoter depends on type of RNA polymerase, more difficult to recognize
Promoters are bound by specific TF’s
Also involves regulatory sequences

43. Transcription in detail
3 steps:
Initiation – RNA polymerase breaks H bonds between DNA pairs and opens up the DNA to form a “transcription bubble”
One strand to be copied is the antisense strand of DNA.
Transcription factors help with this.
Elongation – RNA polymerase adds RNA nucleotides in the 5’
to 3’ direction. The elongation step has a proof-reading
mechanism.
Termination – formation of hair-pin loops of DNA. H bonds
between DNA and RNA are broken and DNA rewinds.

45. Antisense strands vs Sense Strands in DNA Antisense also referred to as template strands; The Sense Strand looks like the mRNA except for the U and T change.

47. Transcription: DNA-> RNA In E
Transcription: DNA-> RNA In E. coli it is possible to see the strands of. RNA transcripts under the electron microscope

48. Miller called these Christmas Trees.
The RNA polymerase molecules and their associated transcripts are so densely packed that the transcripts fan out perpendicularly from the DNA to give each transcription unit a Christmas tree appearance.
The tip of each of these trees is the point on the DNA at which transcription starts and, where the transcripts axe shortest, while the other end is the termination site of transcription and transcripts are longest.

49. The tip of each of these trees is the point on the DNA at which transcription starts and, where the transcripts axe shortest, while the other end is the termination site of transcription and transcripts are longest

50. Pre-messenger Processing
mRNA is further processed in eukaryotes.
Splicing – remove introns, keep exons, job of spliceosomes
5’ cap to provide stability – 7methylguanylate cap
3-end – polyadenylation, a poly A tail which is a stretch of adenine bases. Required for export out of the nucleus as well as stability.

52. Job of spliceosomes

53. Translation in detail 1. Activation:
The 5’ end of the mRNA transcript will bind to a ribosome on the outer membrane of the rough ER
The ribosome recruits tRNAs to the site. The tRNA anticodons matches the codons on the mRNA.
Aminoacyl tRNA synthetase carries out this reaction.
Small subunit of ribosome attaches first and then large subunit.

54. tRNA
Amino acid will attach here.
Anticodon

55. tRNA synthetase
ATP used for energy

56. Elongation –
Ribosome has three binding sites. EPA
Initial binding at the P site.
Add-ons at the A site.
All move down as one in E site exits.

57. Termination
When stop codon on the mRNA sequence is reached.
A release factor will bind and the complex will dissociate.
Protein will fold into its conformation and enter the ER for post translational modification and packaging.

61. Facts about insulin First protein sequenced
Has an alpha chain and beta chain linked by a C-peptide
Gene that codes for it is INS located on Chromosome 11
Secreted by beta cells of the pancreas
Controls blood sugar

62. Insulin Molecular Structure
Insulin is stabilized by two covalent disulfide bonds that join the B-chain and the A-chain and a 3rd bond in the A-chain.

63. Precursor insulin (inactive) is know as preproinsulin.
The first 24 amino acids make up the ER signal sequence. As the protein is being synthesized, this signal sequence begins to emerge from the ribosome. Other proteins in the cell recognize this peptide and dock the ribosome onto the ER.
As the rest of the protein is synthesized, it is directed into the lumen of the ER. From there, the preproinsulin is cleaved into four pieces as it moves through the ER to the Golgi, and to the cell surface.

64. Preparation of insulin in the cell The C-peptide is cut out of proinsulin to create the final mature insulin.
As proinsulin spontaneously folds into its final 3-D shape in the ER, another protease cuts the protein as two sites: between aa 54 and 55 and between aa 89 and 90. As the C-peptide is released from the folded B-chain and A-chain complex, it floats away and is degraded.

65. Modeling Insulin

66. Diabetes and Genetics Type I: onset in childhood
Insulin-dependent – must rely on insulin injections
autoimmune – immune cells attack beta cells of the pancreas

67. Type I Genetics:
Both an inherited and external triggers (diet, infection)
About 18 regions of the genome linked with risk
Most well studied is the HLA genes that encode immune response proteins
The HLA genes are on chromosome 6 and encode for glycoproteins on the surfaces of most cells (MHC complexes) that identify cells
There many different alleles of the HLA genes – many variants of the MHC complexes for individuals
In Type I, 95% of Caucasians inherit one allele for defective genes but very complicated genetically

68. Type II: > 40 years old Insulin not required for survival
Relative insulin deficient, insulin resistance, may be a glucose transport dysfunction, may be due to obesity
Can be controlled by diet, exercise, and in tough cases, insulin injections

69. Genetics/physiology of Type II
In general population, only about half of the people inherit an allele of a defective gene; less than 3% inherit 2 alleles

70. Genetic Engineering of Insulin (1978)

71. 3 stages of cell signaling
Need a review of cell signaling? Campbell Chap 11
3 stages of cell signaling
Reception
Transduction
Response
Boseman video on cell signaling pathways

72. Plasma membrane 1 Reception Receptor Signaling molecule 1
Fig
EXTRACELLULAR
FLUID
CYTOPLASM
Plasma membrane 1 1
Reception
Receptor
Figure 11.6 Overview of cell signaling
Signaling
molecule

73. Plasma membrane 1 Reception Transduction Receptor Signaling molecule 1
Fig
EXTRACELLULAR
FLUID
CYTOPLASM
Plasma membrane 1 1
Reception 2
Transduction
Receptor
Relay molecules in a signal transduction pathway
Figure 11.6 Overview of cell signaling
Signaling
molecule

74. Plasma membrane 1 Reception Transduction Response Receptor Activation
Fig
EXTRACELLULAR
FLUID
CYTOPLASM
Plasma membrane 1
Reception 2
Transduction 3
Response
Receptor
Activation
of cellular
response
Relay molecules in a signal transduction pathway
Figure 11.6 Overview of cell signaling
Signaling
molecule

75. Reception
Ligand – the signal molecule, fits like a lock and key to receptor
Most ligands bind to cell surface receptors; some bind to intracellular receptors
Usually induces a shape change in receptor protein’s shape

76. Types of receptors
Bind with water-soluble (hydrophilic) receptors on membrane:
G-Protein-linked Receptor
Protein Kinase Receptor
Ligand-gated Ion Channel
Bind with hydrophobic receptors:
Intracellular Receptors

77. Protein kinase receptors Tyrosine best understood
Receptor tyrosine kinases (RTK) are enzyme membrane receptors that attach phosphates from ATP to tyrosines (Remember kinase…ATP.)
Once the receptors are activated, relay proteins bind to them and become activated themselves.
A receptor tyrosine kinase can trigger multiple signal transduction pathways at once

78. Fully activated receptor tyrosine kinase
Fig. 11-7c
Signaling
molecule (ligand)
Ligand-binding site
Signaling
molecule
 Helix
Tyr
Tyr
Tyrosines
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Receptor tyrosine
kinase proteins
Dimer
CYTOPLASM 1 2
Activated relay
proteins
Figure 11.7 Membrane receptors—receptor tyrosine kinases
Cellular
response 1
Tyr
Tyr P
Tyr
Tyr P
Tyr
Tyr P P
Tyr
Tyr P
Tyr
Tyr P
Tyr P
Tyr P
Cellular
response 2
Tyr
Tyr P
Tyr
Tyr P
Tyr P
Tyr P 6
ATP
6 ADP
Activated tyrosine
kinase regions
Fully activated receptor
tyrosine kinase
Inactive
relay proteins 3 4

79. Tyrosine Kinase Receptors
Binding of the signal molecules causes the two polypeptides to join.

80. They are activated and act as enzymes to phosphorylate the tyrosines in the tails.

81. The receptor protein is now recognized by relay proteins, triggering different effects.

83. The insulin pathway
Once secreted, insulin binds to its receptor, triggering a cascade of downstream phosphorylation events that expand the initial signal.
1) Insulin binds to its receptor and activates its intrinsic protein tyrosine kinase activity, resulting in the phosphorylation of tyrosine residues located in the cytoplasmic face.
2) The activated receptor, in turn, recruits and phosphorylates a group of substrate molecules which starts a kinase (uses ATP) cascade.
4) That stimulates the glucose transporter GLUT4 vesicles to move to the plasma membrane, resulting in increased glucose uptake.
Insulin action in normal conditions differs depending on the target tissue: glycogen in muscles and live, fatty acids and pyruvate in fat tissue.

84. Insulin Signal Transduction Pathway
Problems here in T2
Diabetes
Type 2 diabetes is accompanied by impaired insulin signal transduction.

85. How is this pathway altered in Type 2 diabetes?
Skeletal muscle accounts for a large percentage of the whole body glucose uptake and is an important site for insulin resistance in type 2 diabetes. It has also been shown that adipose tissue (fat) in type 2 diabetic subjects was resistant to insulin with respect to glucose transport, and had a decreased insulin receptor kinase activity.

86. How would you check for this?
By infusion of glucose and insulin and concomitant monitoring of insulin and glucose levels over several hours, allowing evaluation of peripheral insulin sensitivity, and hepatic glucose production if the infused glucose is labelled either with radioactive or stable isotopes.
Additionally, this study design can be accompanied by obtaining muscle/adipose tissue biopsies, to study the biochemical/physiological effects of insulin and glucose in the peripheral tissues of patients.

87. How insulin signaling works
INSULIN

88. How important is the Glu-4 transporters

89. The Genetic Landscape of Diabetes