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A Reference Guide to Protein Synthesis in Cells

http://www6.slac.stanford.edu/news/2012-12-10-hpylori.aspx

By Katelyn Brandt

Audience and Purpose


This document outlines the process of protein synthesis. The target audience consists of Biochemistry and Molecular Biology students at all levels. For entry level students, this document will act as a guide through the process of protein synthesis, outlining the main events and their order. For those with a greater in-depth knowledge, the document acts as a refresher. The document does not serve as a detailed description, but rather a general overview. The document contains details at the level most professors expect a student to know upon entering their class.

Introduction
Welcome to the world of Biochemistry and Molecular Biology! Four intense (possibly five) years await you. You will sweat, bleed, and break down regularly as you attempt to meet all of your requirements. But dont fret! You should only feel concerned when you do not feel overwhelmed; students who do not feel overwhelmed usually dont understand what the major expects of them.

http://www.mcb.ucdavis.edu/images/advising/bmb/biochemistry.jpg

In this major you will study every chemical and biological process occurring in the cell, and then some. One process you will study extensively and continuously is protein synthesis, the biochemical process of making molecules such as enzymes in the cell. By the time you graduate, you will know this process enough to describe it in great detail. After all, almost every book you buy will include a great in-depth description of the processRight, didnt read them either. So here is a quick reference for you. Keep this in your planner and have the process at your fingertips for every class. This document breaks down the synthesis pathway into its major parts. It also includes enough details to convince your teacher you remember what you learned. Use this as an easy efficient way to jog your memory so you may impress your teachers, convince your parents youve learned something, or annoy your friends.

Table of Contents I. II. III. IV. Quick Glance Step One: Initiation Step Two: Transcription Step Three: Eukaryote Modifications a. 5 capping b. Splicing c. Poly-A tail Step Four: Translation a. Ribosome b. Open Reading Frame c. Amino Acids Step Five: Folding Conclusion

V.

VI. VII.

Quick Glance
Protein synthesis is the biochemical process in the cell involving the transcription and translation of DNA in order to create a biological molecule composed of amino acids. The major steps that occur during protein synthesis include: 1. Translation initiation begins when RNA polymerase (RNAP) binding to the DNA promoter. 2. RNAP moves through the transcription bubble, elongating the messenger RNA (mRNA) transcript. 3. After elongation, mRNA undergoes 5capping, poly-A tail, and splicing in eukaryotes. 4. The mRNA leaves the nucleus and binds to a ribosome. 5. The ribosome adds transfer RNAs (tRNAs) -with attached amino acids- to a growing strand. 6. The strand or primary sequence then dissociates from the ribosome and folds into the proper conformation. 7. Final modifications may be needed to activate the protein.

Step One: Transcription Initiation


The binding of RNAP to the DNA nonsense strand catalyzes initiation. This binding causes a transcription bubble to open, separating the template strand from the non-template strand. This provides the necessary room for RNAP to travel along the DNA. The RNAP will move in the 3-5 direction on the DNA strand, synthesizing mRNA in the 5-3 direction.

The nonsense strand, also considered the bottom strand of the double helix, provides the template RNAP uses to code the mRNA. This gives the nonsense strand the alternate name, the template strand.

http://limbiclab.files.wordpress.com/2012/12/limbic_lab_dna_transcription_diagram1.png

Step Two: Transcription


The RNAP carries out the process of transcription. The RNAP has a conformation known as the right-hand conformation. This provides an area for the RNAP to actively transcribe RNA. During the process, a hybrid strand of DNA and RNA is present in RNAP. Synthesis and proofreading both occur in the single active site of RNAP.

Remember: DNA consists of the bases Guanine, Adenine, Cysteine, and Thymine. RNA uses Uracil instead of Thymine.

http://www.nature.com/nrmicro/journal/v11/n10/images/nrmicro3124-f2.jpg

Transcription terminates according to the DNA strand. Certain sequences or DNA conformations will cause the RNAP to dissociate, ending the transcription process.

Step Three: Eukaryote Modifications


Three main modifications take place in eukaryotes: 5 capping, splicing, and poly-A tail.

5 Capping
The 5 cap consists of adding a methylated guanine in a 5-5 connection. The cap then recruits the splicing machinery.

Splicing
In eukaryotes, genes consist of a series of introns and exons. The final protein product includes only exons. The splicing machinery cuts out the introns to give a series of exons.

Alternate splicing marks a hallmark in higher organism evolution. Scientists discovered introns often include regulatory regions. This differs from the original identification of introns as junk DNA. A gene may encode ten exons, but the protein only needs three. Introns, along with other regulatory factors, tell the splicing machinery which exons to include and which to cut out. But why do genes code for an excess of exons? Because splicing the gene in a different way can give rise to a new protein. This provides a greater complexity to an organism without increasing the genomic size.

http://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/MolBioReview/images/alternative_splicing.gif

Poly-A Tail
The addition of a poly-A tail, a crucial step, allows the mRNA to survive outside the nucleus. Once transcription ends, Poly-A polymerase binds to the mRNA and adds about two hundred adenine bases without a template. This poly-A tail protects the mRNA from proteases in the cytoplasm. The tail is chewed long before the message, giving the mRNA time to find a ribosome and begin translation.

Step Four: Translation


Translation occurs after transcription in the cytoplasm of the cell. The mRNA binds to the ribosome where its code is read and amino acids are linked to form the basic structure of the protein.

The Ribosome
Translation takes place in a ribosome. The mRNA attaches itself to the small ribosomal unit. This signals a large ribosomal unit to bind to the complex, forming the necessary conformation for translation. This conformation, pictured as a hand, includes four sites or fingers: E, P, A, and the thumb.

A: The initial binding site for tRNA as it brings the amino acid. P: The binding site for peptidyl tRNA as it adds its amino acid to the growing chain. E: The exit site for tRNA as its function comes to completion. Thumb: The proofreading site.

http://blogs.scientificamerican.com/scicurious-brain/2013/05/01/scicurious-guest-writer-ribosomes-prepare-to-betranslated/

Open Reading Frame


The ribosome reads the mRNA three base pairs, or a codon, at a time. An open reading frame consists of non-overlapping codons. Each codon has a complimentary anti-codon whose base pairs complement those found in the codon. The tRNA carries both the anti-codon and its associated amino acid on opposite sides of the molecule.

The start codon, AUG usually, associates with a methionine amino acid. The stop codons, UAA, UAG, UGA, simply terminate translation.

http://www.lucasbrouwers.nl/blog/2010/04/evolving-a-code-a-molecular-fossils-tale/

Amino Acids
The world consists of twenty total amino acids. Since four possible bases make up three base segments or codons, sixtyfour different codons can be found in the cell, theoretically matching with sixty-four amino acids. The cell handles this through a process known as degeneracy or the wobble theory. When a tRNA binds to a ribosome, the first two bases of the anticodon must be exact complements of the first two bases of the codon. The third position can remain uncertain. Because of this, different anti-codons can code for the same amino acid.

Amino acids are organic compounds consisting of an amino group and a carboxyl group. Translation starts at the amino end of the peptide strand (N-terminus) and ends at the carboxyl end (C-terminus). This comes from the 5-3 direction of the mRNA read by the ribosome.

Step Five: Folding


The primary structure of a protein refers to its amino acid, or peptide, chain. It folds into alpha helix and beta barrels to make the secondary structure. After this, the protein folds into its three dimensional shape giving it a tertiary structure. Some proteins consist of a complex of tertiary structures or subdomains. When multiple tertiary structures join together they form the quaternary structure.

http://apbrwww5.apsu.edu/thompsonj/Anatomy%20&%20Physiology/2010/2010%20Exam%20Reviews/Exam%201%20Rev iew/Ch02%20Protiens%20and%20Enzymes.htm

Some proteins need further modifications such as phosphorylation or methylation to become activated. However, the formation of the tertiary or quaternary structure signals the end of protein synthesis.

Conclusion
Congratulations you made it through the synthesis of a protein. Remember, this is just a brief overview. This pathway contains many detailed nuances and differences between prokaryotes and eukaryotes. For more information refer to your book The Molecular Biology of the Cell. But that can wait until tomorrow since you most likely need to struggle through a lab report, organic chemistry, or physical chemistry tonight. Best of luck!

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