DNA Structure, Replication, Expression and Regulation

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The Joining of Amino Acids and tRNA

1. The enzyme Aminoacyl-tRNA synthetases fits a unique combination of Amino Acid and tRNA. It begins by pairing the correct anticodon of the tRNA to the codon of the enzyme. The correct amino acid is also linked to the enzyme.

2. Hydrolysis of ATP occurs so the amino acid and tRNA form a covalent bond. Once bonded this is referred to as charged tRNA.

3. The Aminoacyl tRNA synthase releases the charged tRNA to be used in translation.

double helix

antiparallel strands

leading strands only only require one primer while the lagging strand requires multiple primers creating okazaki fragments (DNA polymarase III)

DNA polymarase I removes the RNA primers and replaces with DNA nucleotides

DNA ligase seals the gaps by connecting nucleotides by phosphodiester linkages.

Termination

1. A protein shaped like a tRNA, known as the release factor, is accepted into A site when the stop codon of the mRNA is reached.

2. This protein causes hydrolysis between the last amino acid and the tRNA in P site and the two separate, releasing the polypeptide from the ribosome.

3. The ribosome falls apart. All components of the ribosome separate from one another through the hydrolysis of two GTP.

Elongation

1. Codon Recognition - A charged tRNA pairs it's anticodon with the mRNA codon currently in the A site of the ribosome. Hydrolysis of GTP makes this process more accurate.

2. Peptide Bond Formation - The rRNA in the large ribosomal subunit forms a peptide bond between the carboxyl end of the peptide chain and the new amino acid in A site. In doing this the amino acid and tRNA on P site disconnect and the tRNA is now empty.

3. Translocation - The empty tRNA moves to the E site and is released from the ribosome. The tRNA connected to the peptide chain is moved to the P site (the mRNA is shifted as well) and the A site is empty. The energy from GTP hydrolysis is used here. The cycle is then ready to be repeated.

Initiation

1. The small ribosomal subunit binds to the 5' end of the mRNA made in Transcription once it enters the cytoplasm.

2. The initiator tRNA pairs with the start codon on the mRNA.

3. The large ribosomal subunit attaches itself and the translation initiation complex is formed (this is due to the hydrolysis of GTP). When attached the tRNA is placed in the P site where the peptide chain can begin at the N-terminus with the amino acid MET.

Messleson and Stahl's experiment proved that the correct model of DNA replication is the semiconservative model

The ribosomal subunits are both made of rRNA. The small ribosomal subunit consists of an mRNA binding site. The large ribosomal subunit consists of three tRNA binding sites, consisting of A, P, and E, which the tRNA goes through in that order.

Protein is then shipped to the Golgi through a vesicle

DNA Structure, Replication, Expression and Regulation

Gene Regulation

Gene regulation can occur at any step in gene expression including transcription, mRNA processing, translation, and protein transport.

Transcription is the most critical step for regulating gene expression because gene regulation can block transcription of a certain gene.

Gene Regulation at Transciption

Control elements in DNA bind transcription factors

Types of Control Elements

Distal Control Elements

Sequences in DNA called enhancer sequences that bind specific transcription factors (activators and repressors). Enhancer sequences may be upstream or downstream of a gene and close to or far from the gene they control.

A DNA-bending protein brings the bound activators/repressors closer to the promotor, and RNA polymerase II binds to the promoter, initiating trancription.

Proximal Control Elements

Sequences in DNA close to the promoter that bind general transcription factors

Transcription factors are proteins that bind at the promoter sequence on the DNA strand, increases the binding affinity for the RNA polymerase to recognize the promotor sequence and bind to it, and initiate transcription.

Types of Transcription Factors

Specific

Changes level of transcription

Types of Specific Transcription Factors

Repressors

Reduce levels of transcription down to background level

Activators

Increase levels of transcription above background level

General

Bring about low levels of transcription (background/basal)

Cell specific transcription: combinatorial control of gene expression to increase or decrease expression of different genes

All cells in the body have the same DNA and same genes. Differences in function between cell types result from differential gene expression, the expression of different genes by cells with the same genome.

In prokaryotes, transcription and translation are coupled processes and occur in the cytoplasm because there is no nucleus.

Gene regulation occurs at the level of transcription.

Operons are a cluster of functionally related genes that can be under coordinated control of a single on-off “switch”. The "switch" is a segment of DNA called an operator which is usually positioned within the promoter.

The Lac operon uses both activators and repressors and is an example of both positive and negative regulation.

Glucose in bacterial cell

Adenyl cyclase active

cAMP levels high

CAP active

Adenyl cyclase inactive

cAMP levels low

CAP inactive

Lactose in bacterial cell

Not present

Repressor bound to operator

Lac operon is OFF

Present

Repressor bound to allolactose

Lac operon is ON

Types of Gene Regulation in Prokaryotes

Negative Regulation

Repressor proteins bind operator sequences to decrease expression down to basal level or stop transcription of a gene

Operon is OFF

Positive Regulation

Activator proteins bind operator sequences to increase expression above the basal level

Operon is ON

DNA

REPLICATION

process

primase makes RNA primers complementary to the DNA parent strand sequence

then, DNA polymerase III will add nucleotides only to the 3' end

replication bubble is made at the Origin of Replication (ORI)

Topoisomerase relieve the tension caused by the unwinding of the DNA

Single-Stranded proteins keep the DNA from coming back together

helicase separates the two strands by breaking the hydrogen bonds between the strands

three proposed models of DNA replication

dispersive model

each strand contains parts of both old and newly synthesized DNA

semi-conservative model

the parental strands separate and makes its own new complimentary strand

conservative model

the two parental strands are used as templates, they stay together

STRUCTURE

nitrogenous bases (hydrogen bonding) make up nucleotides

guanine

cytosine

thymine

Adenine

sugar-phosphate backbone

connected by phosphodiester bonds

Translation

Takes place immediately after Transcription since both take place in the cytoplasm here. The mRNA is still translated into polypeptides here through the same steps.

Transcription

3 STAGES

Termination: RNA transcript is released. Polymerase detaches from the DNA

Elongation: New RNA nucleotides are added to the 3' end

Initiation: Start of transcription at +1

Prokaryotes

Location: Cytoplasm

Forms: mRNA

Initiation Enzyme: RNA Polymerase

Eukaryotes

Location: Nucleus

Forms: pre-mRNA then mRNA thru RNA Processing

Initiation Enzyme: RNA Polymerase II

Needs TRANSCRIPTION FACTORS that bind near the promoter before RNA Poly II can bind. TF recognize the TATA box in promoter sequence.

RNA Poly II binds to the promoter upstream the start site

TF + RNA Poly come together to form the translation initiation complex

Termination: 5' cap and 3' Poly A tail

Termination Enzymes: Ribonuclease: Cleavage PolyA polymerase: adds poly A tail, Uses ATP

RNA Processing: Removing of introns and the joining of exons

Spliceosomes: cut out the introns

Protein Transport

Takes secretory pathways. The path taken by protein in a cell on synthesis to modification and then release out of the cell.

Destination of synthesis completed on free ribosomes

All protein synthesis starts on free ribosomes.

Endomembrane System

Polypeptide synthesis begins on free ribosome in cytosol

Synthesis stops temporarily from SRP binding to signal peptide

SRP is signal recognition particle that is made of RNA and protein.

SRP binds to receptor protein in ER membrane

SRP leaves, polypeptide synthesis resumes

Signal peptide is removed by an enzyme in receptor protein complex

Signal peptidase is the enzyme used to cleave the signal peptide.

Protein synthesis finishes inside the ER

Cytoplasm

Organelles

Nucleus

Peroxisomes

Chloroplast

Mitochondria