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Lecture 3 What is a gene, how is it regulated, how is RNA made and processed, how are proteins made...
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Transcript of Lecture 3 What is a gene, how is it regulated, how is RNA made and processed, how are proteins made...
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Lecture 3
What is a gene, how is it regulated, how is RNA made and processed,
how are proteins made and what are their structures?
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How does RNA fit in; its complementary to DNA
Heat
T2 phage dsDNA
ssDNAT2 phage RNA
+
DNA/RNA hybrids
DNA/DNA DNA/RNA RNA
densitygradients
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How do we know DNA makes RNA
70S heavy
30/50S heav y
10 20 30 40 50 60 70 80
14C uracil-labeled RNA
120
100
80
60
40
20
Fraction number
70S heavy
30/50S heav y
10 20 30 40 50 60 70 80
14C uracil-labeled RNA
120
100
80
60
40
20
Fraction number
70S heavy
30/50S heav y
10 20 30 40 50 60 70 80
14C uracil-labeled RNA
120
100
80
60
40
20
Fraction number
5 min pulse lableing 5 min chase 16 min chase
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The simple bacterium E. coli
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Its genome is composed of approximately 5 million base pairs of DNA
DNA
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Within its genome are a few thousand genes
A gene
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Defining a geneWithin a bacterial gene, the information for a protein is found
in a continuous sequence, Beginning with ATG and ending with a STOP codon
5’5’3’
3’
ATGCCGGTACCTCTGAAT...
Met-Pro-Va-Pro-Leu-Asn
AGGGCTGGCCCCTAG
Arg-Ala-Gly-Pro STOP
Start Stop
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Open reading framecoding region
…but what defines a gene?
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Open reading framecoding region
ATG initiationsequence
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Open reading framecoding region
Stop signal
ATG initiationsequence
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How we understand how genes work
• Jacob and Monod defined the lac operon
• Brenner and others determined that an unstable RNA molecule (messanger RNA) was the intermediary between DNA and protein.
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Jacob and Monod made many mutants that could not live on
lactose• These were two types
– those that could be complemented by another wild type gene
– and those that could not be complemented by the presence of a wild type gene
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In the presence of glucose lactose cannot enter the cell
…but deprive the cells of glucose and lactose pours in
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-galactosidase-galactosidase converts lactose to glucose
permease
Permease transports lactose into cells
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z gene y gene
z gene y gene
Mutations in the z or y gene could be complemented by the presence of a second wild type gene. These gene
products are described as working in trans.
The z gene codes for b-galactosidase and y codes for permease
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z gene
y gene
p gene y gene
z gene
p gene
However, mutations in the gene that they called p could not be complemented by the presence of a wild type gene
and so these mutants are know as cis-acting.
What J and M had done was discovered a promoter. A sequencewithin a gene that acted to regulate the expression of the gene.
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Lac Operon
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Negative regulation
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Lac on
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Influence of glucose (positive regulation)
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Open reading framecoding region
Stop signalTranscription start site
Open reading framecoding region
Stop signalTranscription start site
ATG initiationsequence
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Open reading framecoding region
Stop signalTranscription start site
Transcription regulatory sequences
ATG initiationsequence
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Open reading framecoding region
Stop signal
Ribosome binding site
Transcription start site
ATG initiationsequence
Transcription regulatory sequences
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How do we access a specific packet of information (a gene) within the entire genome?
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How does RNA Polymerase work?
Reaction mechanism:(NMP)n + NTP (NMP)n+1 PPi
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How do we know where to start transcription?
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Eukaryotic promoters are more complex. The basal promoter refers to those sequences just upstream of the
gene
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How do we know that these are important regions of the promoter?
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Eukaryotic Promoter
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First a complex of proteins assemble at the TATA box including RNA polymerase II. This is the
initiation step of transcription.
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Regulation of transcription
The next step in gene expression is its regulation. This is much more
complicated then initiation and is still far from being completely understood
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Transcription initiation from the TATA box requires upstream transcription factors
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Regulatory proteins binding to the promoter and enhancer regions of are thought to interact by DNA
folding
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Regulation of promoter region
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Globin gene complex
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Adding a 5’ cap
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Termination and polyadenylation
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Pre-mRNA has introns
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The splicing complex recognizes semiconserved sequences
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Introns are removed by a process called splicing
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snRNPs in splicing
• Donor site Branchpoint Acceptor site
• [----Exon----]GU----------------------A-----(Py)n--AG[----Exon----]
• U1 SnRNP recognises donor site by direct base pairing.• U2 SnRNP recognises branch point by direct base pairing
and subsequently also base pairs at the 5' end of the intron.• U4&U6 (base paired together) are both in same SnRNP.
U6 base pairs with U2. • U5 SnRNP interacts with both donor and acceptor sites by
base pairing, bringing together 2 exons.
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Complexity of genes
• Splicing in some genes seems straightforward such as globin
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For other genes splicing is much more complex
• Fibrillin is a protein that is part of connective tissue. Mutations in it are associated with Marfan Syndrome (long limbs, crowned teeth elastic joints, heart problems and spinal column deformities. The protein is 3500 aa, and the gene is 110 kb long made up of 65 introns.
• Titin has 175 introns.• With these large complex genes it
is difficult to identify all of the exons and introns.
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Alternative RNA splicing
• Shortly after the discovery of splicing came the realization that the exons in some genes were not utilized in the same way in every cell or stage of development. In other words exons could be skipped or added. This means that variations of a protein (called isoforms) can be produced from the same gene.
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1
2 3
41 3
4
4
1
1 2 4
smooth muscleskeletal muscle
PTB binding
polypyrimidine tracts
URE
-tropomyosin pre-mRNA
Alternative splicing of a tropomyosin
There are 3 forms of polypyrimiding tract binding protein (PTB) PTB1, PTB2 and PTB4. Binding of PTB4 to the polypyrimidine suppresses splicing while binding of PTB1
promotes splicing. In smooth muscle exon 3 of a-tropomyosin is not present. Thus, PTB4 is expressed in smooth muscle while PTB1 is not.
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Proteins and Enzymes
The structure of proteins
How proteins functions
Proteins as enzymes
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The R group gives and amino acid its uniquecharacter
Dissociation constants
HA H+ + A-
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Titration curve of a weak acid
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Titration curve of glycine
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Properties of Amino Acids
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Alaphatic amino acidsonly carbon and hydrogen in side group
Honorary member
Strictly speaking, aliphatic implies that the protein side chain contains only carbon or hydrogen atoms. However, it is convenient to consider Methionine in this category. Although its side-chain contains a sulphur atom, it is largely non-reactive, meaning that Methionine effectively substitutes well with the true aliphatic amino acids.
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Aromatic Amino Acids
A side chain is aromatic when it contains an aromatic ring system. The strict definition has to do with the number of electrons contained within the ring. Generally, aromatic ring systems are planar, and electons are shared over the whole ring structure.
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Amino acids with C-beta branching
Whereas most amino acids contain only one non-hydrogen substituent attached to their C-betacarbon, C-beta branched amino acids contain two (two carbons in Valine or Isoleucine; one carbon and one oxygen in Theronine) . This means that there is a lot more bulkiness near to theprotein backbone, and thus means that these amino acids are more restricted in the conformationsthe main-chain can adopt. Perhaps the most pronounced effect of this is that it is more difficult for these amino acids to adopt an alpha-helical conformation, though it is easy and evenpreferred for them to lie within beta-sheets.
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Charged Amino AcidsNegatively charged Positively charged
It is false to presume that Histidine is always protonated attypical pHs. The side chain has a pKa of approximately 6.5, which means that only about 10% of of the species will be protonated. Of course, the precise pKa of an amino acid dependson the local environment.
Partial positive charge
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Polar amino acids
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Somewhat polar amino acids
Polar amino acids are those with side-chains that prefer to reside in an aqueous (i.e. water) environment. For this reason, one generally finds these amino acids exposed on the surface of a protein.
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Amino acids overlap in properties
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How to think about amino acids• Substitutions: Alanine generally prefers to substitute with
other small amino acid, Pro, Gly, Ser.• Role in structure: Alanine is arguably the most boring
amino acid. It is not particularly hydrophobic and is non-polar. However, it contains a normal C-beta carbon, meaning that it is generally as hindered as other amino acids with respect to the conforomations that the backbone can adopt. For this reason, it is not surprising to see Alanine present in just about all non-critical protein contexts.
• Role in function: The Alanine side chain is very non-reactive, and is thus rarely directly involved in protein function. However it can play a role in substrate recognition or specificity, particularly in interactions with other non-reactive atoms such as carbon.
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Tyrosine• Substitutions: As Tyrosine is an aromatic, partially hydrophobic,
amino acid, it prefers substitution with other amino acids of the same type (see above). It particularly prefers to exchange with Phenylalanine, which differs only in that it lacks the hydroxyl group in the ortho position on the benzene ring.
• Role in function: Unlike the very similar Phenylalanine, Tyrosine contains a reactive hydroxyl group, thus making it much more likely to be involved in interactions with non protein atoms. Like other aromatic amino acids, Tyrosine can be involved in interactions with non-protein ligands that themselves contain aromatic groups via stacking interactions.
• A common role for Tyrosines (and Serines and Threonines) within intracellular proteins is phosphorylation. Protein kinases frequently attach phosphates to Tyrosines in order to fascilitate the signal transduction process. Note that in this context, Tyrosine will rarely substitute for Serine or Threonine, since the enzymes that catalyse the reactions (i.e. the protein kinases) are highly specific (i.e. Tyrosine kinases generally do not work on Serines/Threonines and vice versa)
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Cysteine• Substitutions: Cysteine shows no preference generally for substituting
with any other amino acid, though it can tolerate substitutions with other small amino acids. Largely the above preferences can be accounted for by the extremely varied roles that Cysteines play in proteins (see below). The substitutions preferences shown above are derived by analysis of all Cysteines, in all contexts, meaning that what are really quite varied preferences are averaged and blurred; the result being quite meaningless.
• Role in structure: The role of Cysteines in structure is very dependent on the cellular location of the protein in which they are contained. Within extracellular proteins, cysteines are frequently involved in disulphide bonds, where pairs of cysteines are oxidised to form a covalent bond. These bonds serve mostly to stabilise the protein structure, and the structure of many extracellular proteins is almost entirely determined by the topology of multiple disulphide bonds
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Cystine andGlutathione
Glutathione (GSH) is a tripeptide composed of g-glutamate, cysteine and glycine. The sulfhydryl side chains of the cysteine residues of two glutathione molecules form a disulfide bond (GSSG) during the course of being oxidized in reactions with various oxides and peroxides in cells. Reduction of GSSG to two moles of GSH is the function of glutathione reductase, an enzyme that requires coupled oxidation of NADPH.
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Glutamic acid
Histidine
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The peptide bond
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There is free rotation about the peptide bond
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Proteins secondary structure, alpha helix
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Secondary structure, beta pleated sheet
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How enzymes work
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Lock and key
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Specific interactions at active site
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Enzymes lower the energy of activation
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How chymotrypsin works