medicals and genetics...in prokaryotes and eukaryotes, the basics of elongation are the same. •the...

Medical Biology and Genetics VIII

Asst. Prof. N. Ozan TİRYAKİOĞLU

Ph.D.

N. Ozan Tiryakioğlu

Translation

Translation is the process in which ribosomes in the cytoplasm or ER synthesize proteins after the process of transcription of DNA to RNA in the cell's nucleus.


Steps of Translation

• As with mRNA synthesis, protein synthesis can be divided into three phases: initiation, elongation, and termination.

• The process of translation is similar in prokaryotes and eukaryotes.


Initiation of Translation

• Translation begins with the formation of an initiation complex, comprising mRNA, the 40S small ribosomal subunit, Initiation Factors, and nucleoside triphosphates (GTP and ATP).

• The eukaryotic initiation complex recognizes the 7-methylguanosine cap at the 5′ end of the mRNA.


• A cap-binding protein (CBP) and several other IFs assist the movement of the ribosome to the 5′ cap. Once at the cap, the initiation complex tracks along the mRNA in the 5′ to 3′ direction, searching for the AUG start codon.


In prokaryotes and eukaryotes, the basics of elongation are the same.

• The 50S ribosomal subunit of E. coli consists of three

compartments: the A (aminoacyl) site binds incoming charged aminoacyl tRNAs.

• The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA.

• The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids.


• Elongation proceeds with charged tRNAs entering the A site and then shifting to the P site followed by the E site with each single-codon “step” of the ribosome.

• Ribosomal steps are induced by conformational changes that advance the ribosome by three bases in the 3′ direction.

• The energy for each step of the ribosome is donated by an elongation factor that hydrolyzes GTP.


• Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA.

• The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-based enzyme that is integrated into the 50S ribosomal subunit.

• The energy for each peptide bond formation is derived from GTP hydrolysis, which is catalyzed by a separate elongation factor.

• The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain.


• As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled.

• Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200-amino acid protein can be translated in just 10 seconds.


• Termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered.

• Upon aligning with the A site, these nonsense codons are recognized by release factors that instruct peptidyl transferase to add a water molecule to the carboxyl end of the P-site amino acid.

• This reaction forces the P-site amino acid to detach from its tRNA, and the newly made protein is released.


Protein Modifications

• After translation, individual amino acids may be chemically modified, signal sequences may be appended, and the new protein “folds” into a distinct three-dimensional structure as a result of intramolecular interactions.

• A signal sequence is a short tail of amino acids that directs a protein to a specific cellular compartment.


• These sequences at the amino end or the carboxyl end of the protein can be thought of as the protein’s “train ticket” to its ultimate destination.

• Other cellular factors recognize each signal sequence and help transport the protein from the cytoplasm to its correct compartment.


Chemical Modifications, Protein Activity, and Longevity

• Proteins can be chemically modified with the addition of groups including methyl, phosphate, acetyl, and ubiquitin groups.

• The addition or removal of these groups from proteins regulates their activity or the length of time they exist in the cell.


• These changes can alter epigenetic accessibility, transcription, mRNA stability, or translation—all resulting in changes in expression of various genes.

• This is an efficient way for the cell to rapidly change the levels of specific proteins in response to the environment.


• Because proteins are involved in every stage of gene regulation, the phosphorylation of a protein;

– can alter accessibility to the chromosome,

– can alter translation (by altering transcription factor binding or function)

– can change nuclear shuttling (by influencing modifications to the nuclear pore complex)

– can alter RNA stability,

– can modify translation


• The addition of an ubiquitin group to a protein marks that protein for degradation. Ubiquitin acts like a flag indicating that the protein lifespan is complete.

• These proteins are moved to the proteasome, an organelle that functions to remove proteins, to be degraded.

• One way to control gene expression is to alter the longevity of the protein.


Regulation of Gene Expression

• Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product.

• Transcription + Translation


• All cells control or regulate the synthesis of proteins from information encoded in their DNA.

• The process of turning on a gene to produce RNA and protein is called gene expression.


• The regulation of gene expression conserves energy and space.

• It would require a significant amount of energy for an organism to express every gene at all times, so it is more energy efficient to turn on the genes only when they are required


Gene regulation makes cells different

• Gene regulation is how a cell controls which genes, out of the many genes in its genome, are “turned on” (expressed).

• Each cell type in your body has a different set of active genes.

• These different patterns of gene expression cause your various cell types to have different sets of proteins, making each cell type uniquely specialized to do its job.


• For example, one of the jobs of the liver is to remove toxic substances like alcohol from the bloodstream.

• To do this, liver cells express genes encoding an enzyme called alcohol dehydrogenase.

• This enzyme breaks alcohol down into a non-toxic molecule.

• The neurons in a person’s brain don’t remove toxins from the body, so they keep these genes unexpressed, or “turned off.”

• Similarly, the cells of the liver don’t send signals using neurotransmitters, so they keep neurotransmitter genes turned off


How do cells “decide” which genes to turn on?

• As an example, let’s consider how cells respond to growth factors.

• A growth factor is a chemical signal from a neighboring cell that instructs a target cell to grow and divide.

• We could say that the cell “notices” the growth factor and “decides” to divide, but how do these processes actually occur?


EXPRESSION OF GENES

• Gene regulation is the process of controlling which genes in a cell’s DNA are expressed.

• Different cells in a multicellular organism may express very different sets of genes, even though they contain the same DNA.

• The set of genes expressed in a cell determines the set of proteins and functional RNAs it contains, giving it its unique properties.

• In eukaryotes like humans, gene expression involves many steps, and gene regulation can occur at any of these steps. However, many genes are regulated primarily at the level of transcription.


EUKARYOTIC EPIGENETIC GENE REGULATION

• In eukaryotic cells, the first stage of gene expression control occurs at the epigenetic level.

• Epigenetic mechanisms control access to the chromosomal region to allow genes to be turned on or off.

• These mechanisms control how DNA is packed into the nucleus by regulating how tightly the DNA is wound around histone proteins. The addition or removal of chemical modifications (or flags) to histone proteins or DNA signals to the cell to open or close a chromosomal region.

• Therefore, eukaryotic cells can control whether a gene is expressed by controlling accessibility to transcription factors and the binding of RNA polymerase to initiate transcription.


• To start transcription, general transcription factors, such as TFIID, TFIIH, and others, must first bind to the TATA box and recruit RNA polymerase to that location.

• The binding of additional regulatory transcription factors to cis-acting elements will either increase or prevent transcription.

• In addition to promoter sequences, enhancer regions help augment transcription.

• Enhancers can be upstream, downstream, within a gene itself, or on other chromosomes. Transcription factors bind to enhancer regions to increase or prevent transcription.


• Post-transcriptional control can occur at any stage after transcription, including RNA splicing, nuclear shuttling, and RNA stability.

• Once RNA is transcribed, it must be processed to create a mature RNA that is ready to be translated.


• RNA is created and spliced in the nucleus, but needs to be transported to the cytoplasm to be translated.

• RNA is transported to the cytoplasm through the nuclear pore complex.

• Once the RNA is in the cytoplasm, the length of time it resides there before being degraded, called RNA stability, can also be altered to control the overall amount of protein that is synthesized.


• The RNA stability can be increased, leading to longer residency time in the cytoplasm, or decreased, leading to shortened time and less protein synthesis.

• RNA stability is controlled by RNA-binding proteins (RPBs) and microRNAs (miRNAs).

• These RPBs and miRNAs bind to the 5′ UTR or the 3′ UTR of the RNA to increase or decrease RNA stability.

• Depending on the RBP, the stability can be increased or decreased significantly; however, miRNAs always decrease stability and promote decay.


medicals and genetics...in prokaryotes and eukaryotes, the basics of elongation are the same. •the...

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