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BIOL. 2401

Quick Review of Protein Synthesis

Collin County Community College

Proteins are the molecular units that do most of the work in a cell.

They function as molecular catalysts, help in transport, signal transduction, cell recognition, defense mechanism and in various other homeostatic activities.

Proteins and Protein Synthesis

Regulation of protein synthesis is thus of crucial importance for a cell.

Manipulation of these processes or the repair of malfunctioning processes is now possible at the molecular level thanks to the technology of molecular biology and genetic engineering.

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o The genome ( genomic DNA ) contains the genetic information

Protein Synthesis Review

o The specific information that specifies the amino acid sequence in a protein is contained within the linear sequence of the nucleotide bases within a DNA gene

o Each amino acid is represented by 3 nucleotide bases within the DNA (= triplet codon)

o DNA does not participate directly in the making of a protein ; active DNA genes produce messenger RNA which migrate to the cytoplasm where they generate proteins with the help of ribosomes and transfer-RNA

o The process of transferring DNA information into mRNA = transcription

o The process of making a protein from mRNA = translation

Protein Synthesis Review

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DNA : double helix molecule

Nucleotides: (sugar, phosphate, base)

Nucleotide Sequence Information

Triplet Codon

messenger RNA (mRNA) passes the information from DNA to the cytoplasm before a protein can be made

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DNA : double helix molecule

Gene Activation is the process where the information within a gene becomes expressed. That information usually means the production of a protein with a specific function.

Gene activation requires the• uncoiling of DNA to use it• Copying the instructions from DNA to mRNA (known as

Transcription, which occurs in the nucleus)• Transcription involves the RNA polymerase, which produces

messenger RNA (mRNA)

DNA to mRNA : Transcription

• The process of Transcription requires Transcription Factors ; they are a class of proteins that can bind to DNA, the promotor site or to RNA polymerase

• They will start or inhibit the process of transcription• The promotor site is where the RNA polymerase starts “reading”

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Transcription factors

This Transcription Factor Prevents the ‘reading’ of Gene A

This one Activates the ‘reading’ of

Gene B

PostTranscriptional Process

• A mRNA contains DNA information with relevant and non-relevant (nonsense) information

• The portion of DNA with relevant information = EXON• The portion of DNA that is not needed = INTRON• The initial mRNA will contain these transcribed portions

of introns and exons = pre-mRNA

• Splicing is the process in which introns and “spliced”outof the mRNA and exons are joined together

• Splicing is handled by Spliceosomes in the nucleus

• The functional mRNA moves into the cytoplasm through the nuclear pores and into the rough ER

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mRNA Splicing

= removed

Protein Synthesis = Translation

• Once in the cytoplasm, a functional polypeptide is constructed at Ribosomes, using the information in the mRNA codons

• The linear sequence of the codons determines the sequence of amino acids

• Specific Transfer RNA’s (tRNA) bring sequential Amino Acids to the ribosomal complex according to their complementary base pairing of anticodons

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Transfer RNA and Anticodon

Amino Acid

mRNA to Protein : Translation

Certain small proteins known as ribosomal initiation factors are required to establish an " initiation" complex for translation !

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mRNA to Protein : Translation

When ribosomes reach the stop codon, the system falls apart and the protein is complete. In this example, the completed protein has 3 amino acids.

Protein Synthesis

The longer a mRNA, the more ribosomes can fit onto it and read it, the more protein can be made per time unit.

The picture below show plenty of ribosomes reading a mRNA … lots of protein production going on there !

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PostTranslational Process

Once a protein is made, it may not be functional yet. The process of making a protein functional is called PostTranlationalmodification. There are many ways this can happen.

This can include removal of precursor elements or signaling elements.Insulin for example is first created as a pre-pro-insulin protein. Removal of the precursor signaling peptide, results in pro-insulin. Proinsulin is modified and cleaved again, yielding active insulin

Some mRNA’s create proteins that are actually 2 or 3 functional proteins. By cleaving the initial protein, 2 or 3 functional proteins are created.

PostTranslational Process

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The action-effect of a protein depends on its activity and on its concentration.

Concentration is dependent on synthesis, stabilization and degradation and as such, can be affected by modulation of these processes.

[Protein]Synthesis Degradation

Modulation

Stabilization

Overall Control of Protein Activity

Transcription of DNAo Transcription factors can activate or repress the transcription processo Activating or inhibition of transcription factors can thus result in more

or less mRNA

Factors that determine Protein Levels

Splicing of mRNAo The activity of the spliceosomes will determine how many "active"

mRNA will be produced, and thus how functional the protein production will be.

mRNA regulationo RNAses are enzymes that break down mRNA.o Longevity of the mRNA depends on factors that resist the mRNA

from being broken down. o The longer a mRNA molecule exists, the more protein can be

produced from that mRNA.

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Translation of the mRNAo Certain small proteins known as ribosomal initiation factors are

required to establish an " initiation" complex for translation.o The effectiveness of the ribosome initiation complex with the

mRNA determines how effective the protein production will be.

Factors that determine Protein Levels

Protein degradationo Every proteins has a certain life span and is subject to degradationo Proteins are targeted for degradation by the attachment of a small

protein called Ubiquitin.o This protein directs it to a proteasome which breaks down the proteino Proteins can be protected by other proteins that prevent them from

breakdown and unfolding ( e.g. heatshock proteins).

Protein Degradation

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Allosteric and covalent modificationo This process does not change the effective concentration of a proteino It modulates ( = changes) the activity of a protein by inhibiting or

increasing its efficiency

Factors that determine Protein Levels

Allosteric modulation

• Due to a the binding of a modulator molecule at a site away from the active site

• This other binding site is known as the regulatory site• The result of a modulator molecule binding to the regulatory site,

is that it changes the conformation of the active site and thus alters the properties of the active site.

Covalent modification

• The activity of a protein is changed by the covalent bonding of a chemical group to the protein

• Most common way is phosphorylation : it is the attachment of a phosphate group to a protein. The phospate group usually comes from ATP

• Phosphorylation affects the ligand binding properties of the protein.

Factors that determine Protein Levels

• Enzymes that mediate phosphorylation are called protein kinases• Enzymes that remove the phosphate are called (phospho)-protein

phosphatases.

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Protein Kinases & Phosphorylation

A protein Kinase is an enzyme that adds a phosphate group to a protein. By doing so, it can activate (“wake-up”) a dormant protein (such as an enzyme).

A protein Phosphatase is an enzyme that removes the phosphate group from protein. By doing so, it reverses the previous action and puts the enzyme back to ‘sleep’ !

Allosteric Vs. Covalent Modulation

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Factors that determine Protein Levels