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?

Defining a gene
Within a bacterial gene, the information for a protein is found in a continuous sequence, Beginning with ATG and ending with a STOP codon

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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.

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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Lac Operon

Negative regulation

Lac on

Influence of glucose (positive regulation)

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How do we access a specific packet of information (a gene) within the entire genome?

How does RNA Polymerase work?

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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

How do we know that these are important regions of the promoter?

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Eukaryotic Promoter

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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Regulation of promoter region

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Globin gene complex

Adding a 5’ cap

Termination and polyadenylation

Pre-mRNA has introns

The splicing complex recognizes semiconserved sequences

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.

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.

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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Proteins and Enzymes

The structure of proteins

How proteins functions

Proteins as enzymes

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Properties of Amino Acids

Alaphatic amino acids
only carbon and hydrogen in side group

Aromatic Amino Acids

Amino acids with C-beta branching

Charged Amino Acids

Polar amino acids

Somewhat polar amino acids

Amino acids overlap in properties

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.

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)

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


	What is a gene, how is it regulated, how is RNA made and processed, how are proteins made and what are their structures?


	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.


	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


	The next step in gene expression is its regulation. This is much more complicated then initiation and is still far from being completely understood


	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.


	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.


	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.


	The structure of proteins
	How proteins functions
	Proteins as enzymes


	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.


	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)


	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