Chapter 17 From Gene to Protein

Main Questions: The information content in DNA is the specific sequence of nucleotides along the DNA strands. How does this information determine the organism’s appearance? How is the information in the DNA sequence translated by a cell into a specific trait?

The Bridge Between DNA and Protein RNA is the single stranded compound that carries the message from the DNA to the ribosome for translation into protein. Recall, DNA = A,T,C,G; RNA= A,U,C,G The order of these bases carries the code for the protein which is constructed from any or all of the 20 amino acids.

Transcription and Translation Going from gene to protein. Transcription is the synthesis of mRNA using DNA as the template, and is similar to DNA synthesis. mRNA is the message (hence the “m”) from the gene. Translation is the process that occurs when the mRNA reaches the ribosome and protein synthesis occurs.

RNA RNA is used because it is a way to protect the DNA from possible damage. Many copies of RNA can be made from one gene, thus, it allows many copies of a protein to be made simultaneously. Additionally, each RNA transcript can be translated repeatedly--via polyribosome.

Recall the Main Difference Between prokaryotes and eukaryotes, there is one main difference between transcription and translation. The two processes can occur simultaneously in prokaryotes because they lack a nucleus. In eukaryotes, the two processes occur at different times. Transcription occurs in the nucleus, translation occurs in the cytoplasm.

The Genetic Code 61 of the 64 codons code for amino acids. 3 of the codons code for stop codons and signal an end to translation. AUG--start codon

Genetic Code The genetic code is said to be redundant. More than one triplet codes for the same amino acid. One triplet only codes for one amino acid. The reading frame is important because any error in the reading frame codes for gibberish.

Transcription The gene determines the sequence of bases along the length of the mRNA molecule. One of the two regions of the DNA serves as the template. The DNA is read 3’-->5’ so the mRNA can be synthesized 5’-->3’

Translation mRNA triplets are called codons. Codons are written 5’-->3’ Codons are read 5’-->3’ along the mRNA and the appropriate aa is incorporated into the protein according to the codon on the mRNA molecule. As this is done, the protein begins to take shape.

mRNA and RNA Polymerase mRNA is the “messenger” or vehicle that carries the genetic information from the DNA to the protein synthesizing machinery. RNA polymerase pries apart the DNA and joins RNA nucleotides together in the 5’-->3’ direction (adding, again, to the free 3’ end). RNA polymerase is just like DNA polymerase, but it doesn’t need a primer.

The Synthesis of mRNA RNA pol II encounters a promoter on the DNA near a transcriptional unit and starts synthesizing RNA. When the RNA pol II encounters a terminator sequence, transcription stops.

The Synthesis of mRNA RNA pol II encounters a promoter on the DNA near a transcriptional unit and starts synthesizing RNA. When the RNA pol II encounters a terminator sequence, transcription stops.

Different Types of RNA Polymerase Prokaryotes have one type of RNA polymerase that synthesizes mRNA and the other types of RNA as well. Eukarytoes have 3 different types in their nuclei (I, II, III). mRNA synthesis uses RNA pol II.

Promoters Promoters are found on the DNA molecule and initiate the transcription of the gene. This is the site where transcription factors and RNA polymerase attach.

Promoters Transcription is finished when the RNA polymerase reaches the terminator. The stretch of DNA that is transcribed is known as the transcription unit. Promoters serve as great examples of non- coding DNA that has a function.

Promoters Promoters are specific base sequences to which specific transcription factors (proteins) bind to initiate gene expression. They usually extend a few dozen nucleotides upstream from the transcription start point. Include a “TATA box” in eukaryotes. Promoters are important for the binding of the RNA polymerase.

The Initiation of Transcription Transcription factors bind to the promoter region enabling RNA pol II to do so. The RNA pol II binds with additional transcriptional factors creating a transcription initiation complex. DNA unwinds and transcription begins.

RNA pol II

Promoter Differences Between Prokaryotes and Eukaryotes In prokaryotes, RNA polymerase recognizes and binds to the promoter on the DNA associated with sigma factor proteins and immediately begins synthesizing mRNA. In eukaryotes, a group of proteins called transcription factors are needed for the binding of the RNA polymerase and the initiation of transcription.

Promoter Differences Between Prokaryotes and Eukaryotes Once the transcription factors bind to the promoter, RNA pol II binds and transcription can then proceed. The entire group of proteins in the eukaryote are called the transcription initiation complex.

Transcription As the RNA pol II moves along the DNA, it uncoils it, synthesizes the mRNA transcript and peels away from the DNA allowing it to recoil. Numerous RNA polymerases can transcribe the same DNA segment (protein) at the same time. This enables the cell to make large amounts of protein in a short period of time.

Transcription An electron micrograph showing the transcription of 2 genes.

Transcription Termination In prokaryotes transcription proceeds through a DNA sequence that functions as a termination signal causing the disassembly of the transcription complex and the polymerase to detach from the DNA. This release of the transcript makes it immediately available for use as mRNA in prokaryotes.

Transcription Termination In eukaryotes, when the RNA pol II reads a certain signal sequence, it cleaves off the RNA from the growing chain as RNA pol II continues transcribing DNA. The RNA pol II continues to read and transcribe DNA and eventually falls off the DNA template strand, (not fully understood). The RNA produced now is still not ready for use.

RNA Modification The eukaryotic RNA transcript now gets modified before it enters the cytoplasm. The 5’ end of the transcript gets modified before leaving the nucleus--a 5’ cap of nucleotides. The 3’ end is also modified--numerous adenine nucleotides--called a poly-A tail.

Important Functions of the 5’ Cap and Poly-A Tail They facilitate export of the mature mRNA from the nucleus. They protect mRNA from degradation by hydrolytic enzymes. They assist in the attachment of the ribosome to the 5’ end of the mRNA.

mRNA Modification The mRNA is further processed after the ends have been modified--RNA splicing. The initial transcript (~8000 bp) is reduced (to ~1200 on average). The large, non-encoding regions of the DNA that get transcribed are spliced out. Introns--intervening regions are removed. Exons--expressed regions are kept.

mRNA Modification Some untranslated regions of the exons are saved because they have important functions such as ribosome binding. RNA splicing occurs via snRNP’s. snRPs consist of RNA and protein and join together to form a spliceosome which interacts with the intron to clip it out and join the exons together.

So, Why is RNA Splicing Significant? In many genes, different exons encode separate domains of the protein product.

RNA Splicing The way the RNA is spliced determines which proteins will be expressed. The different sexes of some organisms splice RNA differently and thus translate the genes into proteins differently--contributing to differences seen among sexes. The alternative RNA splicing is one possible reason humans can get by with relatively few genes.

Translation Translation is when the cell interprets the genetic message and builds the polypeptide. tRNA acts as the interpreter. tRNA transfers aa’s from the cytoplasm to the ribosome where they are added to the growing polypeptide. All tRNA molecules are different.

tRNA Structure and Function tRNA, like mRNA, is made in the nucleus and is used over and over again. tRNA binds an aa at one end and has an anticodon at the other end. The anticodon acts to base pair with the complementary code on the mRNA molecule, and delivers an aa to the ribosome.

tRNA Structure and Function As tRNA reads the mRNA transcript, it brings an aa to the ribosome and adds it to growing polypeptide. The 2D shape is similar to a cloverleaf.

2 Recognition Steps in Translation 1. There must be a correct match between tRNA and an aa. 2. The accurate translation of the mRNA molecule.

1. The Correct Match 1. Each aa gets joined to a tRNA by aminoacyl-tRNA synthase--there are 20 of these, one for each amino acid. This enzyme catalyzes the attachment of aa to tRNA with the help of some ATP energy. The activated aa is now ready to deliver the aa to the growing polypeptide.

2. Accurate Translation The tRNA must correctly match up the tRNA anticodon with an mRNA codon. There is not a 1:1 ratio of the tRNA molecules with mRNA codons. Some tRNA’s can bind to more than one codon. This versatility is called “wobble.”

2. Accurate Translation Wobble enables tRNA to bind differently in one of its base pairs. This is why codons for some aa’s differ in their 3rd base. For example: the uracil at the 5’ end of a tRNA anticodon can pair with an A or a G in the third position of the 3’ end of the mRNA codon.

Ribosomes These are the sites of protein synthesis. They consist of a large and a small subunit and are comprised of RNA and protein. The RNA is ribosomal RNA (rRNA). Bacterial (70s, 50S + 30s) Eukaryotic (80s, 60S + 40s)

Ribosomes rRNA genes are found on chromosomal DNA and are transcribed and processed in the nucleolus. They are assembled and transferred to the cytoplasm as individual subunits. The large and small subunits form one large subunit when they are attached to the mRNA.

Ribosomes The structure of ribosomes fit their function. They have an mRNA binding site, a P-site, an A-site and an E-site. A-site (aminnoacyl-tRNA) holds the tRNA carrying the next aa to be added to the chain. P-site (peptidyl-tRNA) holds the tRNA carrying the growing peptide chain. E-site is the exit site where the tRNAs leave the ribosome. Each of these are binding sites for the mRNA.

The 3 Stages of Protein Building 1. Initiation 2. Elongation 3. Termination All three stages require factors to help them “go” and GTP to power them.

1. Initiation Initiation brings together mRNA, tRNA and the 2 ribosomal subunits. Initiation factors are required for these things to come together. GTP is the energy source that brings the initiation complex together.

2. Elongation The elongation stage is where aa’s are added one by one to the growing polypeptide chain. Elongation factors are involved in the addition of the aa’s. GTP energy is also spent in this stage.

Recall from Chapter 5 As the amino acids are being joined together, the sequence and number of the amino acids gives the protein its primary structure. 50

Recall from Chapter 5 The secondary structure is forming simultaneously as the hydrogen bonding between the amino acids give a-helicies and ß-pleated sheets. 51

Recall from Chapter 5 The tertiary structure is formed as more amino acids are added and the R-group interactions work to stabilize the protein. 52

Recall from Chapter 5 Lastly, the final functional protein structure forms as multiple polypeptide chains join to give the quaternary structure. Not all proteins exist as multiple polypeptides. 53

3. Termination Termination occurs when a stop codon on the mRNA reaches the “A-site” within the ribosome. Release factor then binds to the stop codon in the “A-site” causing the addition of water to the peptide instead of an aa. This signals the end of translation.

Polypeptide Synthesis As the polypeptide is being synthesized, it usually folds and takes on its 3D structure. Post-translational modifications are often required to make the protein function. Adding fats, sugars, phosphate groups, etc. Removal of certain proteins to make the protein functional. Separately synthesized polypeptides may need to come together to form a functional protein.

Eukaryotic Ribosomes Recall the 2 types: Free and bound. They function exactly the same and can switch from free to bound. This switch can occur when the protein that is being translated contains a signal peptide instructing the ribosome to attach to the ER. Once attached to the ER, synthesis will continue to completion and can then be exported from the cell.

Signal Peptide Recognition The signal peptide is recognized as it emerges from the ribosome by a protein- RNA complex called signal-recognition particle. The particle functions by bringing the ribosome to a receptor protein built into the ER where synthesis continues and the growing peptide finds its way into the lumen.

Signal Peptide Recognition Once in the lumen of the ER, the newly synthesized polypeptide is modified. The signal peptide is cut out by an enzyme. The protein then undergoes further processing and is shipped where it needs to go.

Differences in Prokaryotic and Eukaryotic Gene Expression Prokaryotic and eukaryotic RNA polymerases are different, but perform the same function. Transcription is terminated differently. Prokaryotic and eukaryotic ribosomes are different. Transcription and translation are streamlined in prokaryotes, it is compartmentalized in eukaryotes. Eukaryotic cells have a complex system of targeting proteins for their final destination.