Gene Expression/Regulation

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Structural genes

Genes whose expression is controlled together

Lac A

gene for B-Galactoside transacetylase

Lac Y

Lac Z

gene for B-Galactosidase

Regulatory gene

Lac I

Codes for repressor protein

Regulatory regions

Promoter

Occurs structural and regulatory gene/s

Operator

"switch" is a segment of DNA

location where protein binds

Binding causes positive/negative regulation

activators/repressors

Gene expression turned on

Gene expression turned off

After mRNA processing, it will leave the nucleus where all the chromatin is located. to then move into the cytoplasm and find a ribosome where translation occurs.

Gene Expression/Regulation

Gene Regulation

Activators/repressors are activated/made

far away from the gene

activator bind to mediator proteins

brings the activator closer to the promotion

DNA bending brings activator closer to promotion site

activator binds to enhancer

specific transcription factor

turns gene regulation on/off

last gene that enters the nucleus

repressor

operon gene regulation on

activator

binds to operator

protein

Activators/repressors involvement

Transcription factors

help increase or decrease level of transcription

General

background/basal

low levels of transcription

bind to promoter and regions near

Specific

changes levels of transcription

increase levels of transcription

done by activators

high levels of transcription are reduced by repressors

Bind to distal control elements called enhancers

Present near or far from gene they are controlling

control elements in DNA

proximal

sequences in DNA close to promoter

Bind general transcription factors

distal

sequences in DNA upstream or downstream

can be close or far from gene they are controlling

Bind to specific transcription factors

operons

helps with regulation with an on-off switch

"switch"

segment of DNA known as "operator"

positioned within promoter

proteins bind to operators to turn on gene expression for multiple genes

OR to turn off expression

positive regulation

Prokaryotes

occurs at level of transcription

gene expression on

with activator, transcription occurs

expression at high level

gene expression off

transcription occurs

no transcription occurs

repressor protein bond to operator sequence

Lac operon

regulation needs both repressor and operator

lactose

disaccharide made of glucose and galactose

inducer of the lac operon

cAMP level high

abundant lac mRNA synthesized

operon on: induced/high expression

Activator protein CAP is activated by cAMP

CAP helps RNAP bind to promoter

facilitates transcription

negative regulation

transcription of structural genes is blocked

operon on

mRNA translates: B-Galactosidase, Permease, transacetylase

takes in more lactose from outside

breaks it down to glucose and galactose

uses sugars as needed

all structural genes are transcribed

forms a long mRNA

no glucose = operon on

inducible operon

cAMP level low

operon off

blocks adenylyl cyclase

prevent production of cAMP

CAP cannot be activated

CAP can't help RNAP bind promoter

little mRNA synthesized

Eukaryotes

Regulation through operons

transcription factors (instead of operons)

activators

repressors

Operons do not occur in eukaryotic cells - made in mRNA w/ individual promoter

Gene organization

Distal

enhancers

bind activator proteins

activator bound to receptor is brought to promoter via DNA bending proteins

Transcription increased via RNA polymerase II

Proximal

basal (general) expression

Specific to eukaryotes (ex. humans)

DNA Structure

Physical Structure

DNA is made of two strands that wind around each other. These strands are made of a chain of monomers and the two strands are joined at the base of each monomer. They wind around each other, forming the shape known as a double helix.

Monomer

The monomers of DNA are nucleotides. Nucleotides are made up of three parts: a sugar, a base, and a phosphate.

A phosphate group is a functional group made of one phosphorus atom bonded to four oxygen atoms. Each DNA monomer has one phosphate group attached to it.

A base is a nucleotide found in the "rungs" of the DNA double helix ladder. These bases are connected by hydrogen bonds.

DNA contains a deoxyribose sugar. A deoxyribose sugar is formed by a 5-carbon ring and a hydrogen at the 2' position instead of a hydroxyl group

Nucleotide Bases

Base pairs in DNA connect the backbones of the DNA together. The backbones and the base pairs connecting them form the ladder-like double helix structure of DNA.

These base pairs are joined together by hydrogen bonds. The pairs themselves are able to connect based on their structures. These structures are called purines and pyrimidines.

Purines have an additional ring where pyrimidines do not. Adenine and Guanine are purines and only bond to pyrimidines

A bonds to T G bonds to C

Pyrimidines do not have the same additional ring in their structure that purines do. Thymine and cytosine are pyrimidines and only bond to purines.

The formation of RNA from DNA is an important step in DNA replication. RNA varies from DNA in a few different ways.

Structurally, DNA contains a deoxyribose sugar where RNA contains a ribose sugar, which has a hydroxyl at 2'.

DNA is responsible for the transfer of genetic information where RNA is responsible for transmitting coding proteins for proteins creation.

The main difference between DNA and RNA is that DNA is double stranded where RNA only exists as a single strand.

Replication

Hallmarks of DNA replication: Speed- In E. coli 5 million bases are copied at the rate of 2000 nucleotides per second! Accuracy- DNA replication is high accurate. Processivity- Protein called sliding clamp converts the DNA polymerase III from being distributive (falling off) to processive (staying on).

The first step in DNA replication is to unzip the double helix structure of the DNA molecule. This is carried out by an enzyme called helicase, which breaks the hydrogen bonds holding the complementary bases of DNA together.

Other Enzymes that assist: Single-strand binding protein- the protein that prevents the strands from reforming the hydrogen bonds. Topoisomerase- an enzyme that helps relieve any unwound DNA. Sliding clamp- helps load up the template of parent strand to polymerase.

The separation of the two single strands of DNA creates a Y shape called a replication fork. The two separated strands will act as replates for making the new strands of DNA

One of the strands is oriented in the 3' to 5' direction, this is the leading strand. The other strand is oriented in the 5' to 3' direction, making this is the lagging strand. As a result of their different orientations, the two strands are replicated differently.

Lagging Strand

Numerous RNA primers are made by the primase enzyme and bind at various points along the lagging strand.

Chunks of DNA, called Okazaki fragments, are then added to the lagging strand also in the 5' to 3' direction. This type of replication is called discontinuous as the Okazaki fragments will need to be joined up later.

Leading Strand

A short piece of RNA called a primer, produced by an enzyme called primase, comes along and binds to the end of the leading strand. The primer acts as the starting point for DNA synthesis.

DNA polymerase binds to the leading strand and then goes along it, adding new complementary nucleotide bases to the strand of DNA in the 5' to 3' direction. This sort of replication is called continous.

Once all of the bases are matched up, an enzyme called exonuclease trips away the primers. The gaps where the primers were are then filled by more complementary nucleotides. This new strand is proofread to make sure there are no more mistakes in the new DNA sequence.

Finally, an enzyme called DNA ligase seals up the sequence of DNA into two continuous double strands. The result of DNA replication is two DNA molecules consisting of one new and one old chain of nucleotides. This is why DNA replication is described as semi-conservative, half of the chain is part of the original DNA molecule, half is brand new.

Transcription

In Eukaryotes, transcription now occurs in the nucleus while translation still occurs in the cytoplasm. They are no longer coupled, and there is now pre-mRNA because of RNA processing.

In Prokaryotes, transcription occurs in the cytoplasm, as well as translation. Because these two processes are able to occur together, this means that they are coupled, and can be immediately translated.

First step of Transcription: Initiation (Starts at the "Transcription start point", otherwise known as the first nucleotide or +1.) Initiation is completed by an enzyme known as RNA polymerases, but differs in forms when it comes to prokaryotes and eukaryotes. In prokaryotes, it is simply RNA polymerase, otherwise known as RNAP. RNA polymerases bind to a region on the DNA upstream of the start site, known as the promoter. DNA strands then unwind, and the polymerase initiates RNA synthesis at the start point of the template strand. (To start transcription, RNA polymerases do not need a primer, nor do they need helicases.) In eukaryotes, it is RNA polymerase II that is responsible and forms the pre-mRNA (as well as snRNA, and microRNA). The transcription process for Eukaryotes also requires additional proteins that are called transcription factors. They first bind to the promoter and the region near the promoter before RNA polymerase II can bind and later unwind the DNA strand.

Second step of Transcription: Elongation In the process of elongation, the new RNA nucleotides are added to the 3' end of a growing chain. The new strand can ONLY be replicated in one order, from the 5' to 3' end, so the bottom template strand of 3' to 5' is chosen to make the new 5' to 3' strand of mRNA. After this is completed, the DNA strands re-form a double helix.

Third and Final step of Transcription: Termination Once the RNA polymerase reaches a transcription termination site on the DNA, the RNA transcript is released, and the polymerase detaches from the DNA. Transcription is then stopped, with the completion of a 5' to 3' end RNA strand. However, in Eukaryotes, the termination of transcription is a bit different. A sequence of "AAUAAA" is a signal for the cell to cut the newly formed strand of pre-mRNA and release it from the DNA. At the 5' end of the strand, a modified G nucelotide named CAP is added, and at the 3' end of the strand, near the AAUAAA sequence, a polyA tail is added by the enzyme polyA polymerase. (The 5' CAP will be used for translation and the 3' polyA tail helps with the stability of the mRNA.) These alterations are ONLY found in Eukaryotes. Before we can move on to translation, the pre-mRNA has to go through RNA processing before it can exit the nucleus. This process involves the removal of introns and the joining together of exons.

Translation

Translation can be defined as the formation of proteins from an mRNA template. This process consists of three phases: initiation, elongation, and termination.

The eukaryotic mRNA which is a substrate for translation consists of a unique 3' end named the poly-A tail. This mRNA also has codons that encode for specific amino acids. The 5' end of the tail is a methylated cap.

This process occurs inside a ribosome. The small ribosomal subunit binds to an mRNA cap and moves to the initiator tRNA that connects to the start codon (AUG) with its complimentary anti codon (UAC). Attached to tRNA is methionine that cooresponds to the AUG codon. The large ribosomal subunit then connects as well to complete the translation initiation complex by creating the P and A sites.

The first tRNA occupies the P site as the second tRNA enters the A site as it's complimentary to the second codon. The methionine is transferred to the A-site amino acid. The beginning tRNA detaches itself to leave the ribosome, causing a shift to create space for the next tRNA. This is the process of elongation.

The previous process of exiting tRNA's for movement of the ribosome along the mRNA to allow for the next tRNA continues until a stop codon is reached in the A-site (those being UAG, UAA, AND UGA). A release factor enters the A-site and translation is terminated.

When termination is reaches the ribosome dissociates and the newly formed protein is released

This allows for a polypeptide chain to grow until the stop codon is reached, causing the polypeptide to then float away and enter a cell organelle for folding and further modification.

In this process, the mRNA acts as a code for a certain protein. This is made possible through the bases on mRNA that are called codons which code for specific codons named anticodons (carried by the tRNA strand).

The start codon, AUG, begins translation while stop codons UAA, UAG, and UGA terminate it.