eukaryotic gene expression Flashcards
(21 cards)
Define gene expression.
The process by which the information within a gene is used, first to synthesise RNA (transcription) and then to synthesise a polypeptide (translation) eventually to affect the phenotype of an organism.
Describe the Central Dogma of Molecular Biology.
A two stage process where genetic information proceeds unidirectionally from DNA to RNA (transcription) to polypeptide (translation).
What are the different types of RNA and their functions?
1 Messenger RNA (mRNA) - Carries information, which codes for amino acid sequences, from DNA to ribosomes
2 Transfer RNA (tRNA) - Serves as an adaptor molecule in protein synthesis, Translates mRNA codon sequence into amino acid sequence
3 - Ribosomal RNA (rRNA) - Plays catalytic and structural roles in ribosomes
4 Small nuclear RNA (snRNA) - Plays catalytic and structural roles in spliceosomes, the complexes of protein and RNA that carry out splicing of pre-mRNA
5 Small interfering RNA (siRNA) & microRNA (miRNA) - involved in regulation of gene expression
Define transcription.
The process by which a complementary RNA copy is made under the direction of the template strand of a specific region of the DNA molecule, catalysed by the enzyme RNA polymerase.
Define a gene.
ο· A gene is a section of DNA that encodes information in the form of a specific base sequence to direct the synthesis of one polypeptide chain or RNA molecule.
ο· It is a unit of inheritance located in a fixed position (locus) on the chromosome which specifies a particular character of an organism.
Describe the promoter and its importance.
ο· It contains the TATA box and the transcription start site (+1: the nucleotide where RNA
synthesis begins).
ο· The TATA box serves as a binding site for a general transcription factor, i.e. TFIID. The TATA box is typically located 25 bp upstream of the transcription start site.
ο· The promoter determines which of the two strands of DNA is used as the template.
ο· The promoter is a short nucleotide sequence that is NOT transcribed except for the
transcription start site.
In what direction is the DNA read?
The template DNA strand is read in the 3β to 5β direction to facilitate synthesis of RNA in the 5β to 3β direction.
Describe the steps in initiation of transcription.
- General transcription factors are assembled along promoter, TFIID binds to TATA box. Mediates the binding of RNA polymerase to the promoter, forming transcription initiation complex.
- Binding of RNA polymerase to promoter causes DNA double helix to unwind and separate. Hydrogen bonds between complementary base pairs are disrupted and a transcription bubble exposing a short stretch of nucleotides on each strand is created.
- One of the exposed RNA strands acts as a template for complementary base pairing to directly assembly of incoming ribonucleotides. RNA polymerase catalyses the formation of the first phosphodiester bond.
Describe the steps in elongation of transcription.
- As RNA pol moves along the template DNA, DNA double helix continues to the transiently unwind.
- Ribonucleotides continue to form complementary base pairs with the DNA template; As each ribonucleoside triphosphate is brought in, its two terminal phosphates are removed and the remaining free 5β phosphate group is added to the free 3β OH group of the growing RNA chain via the formation of a phosphodiester bond catalysed by RNA pol.
- RNA pol reanneals the unwound DNA behind it, dissociating the growing RNA chain from the template + carries out proofreading functions, removing any incorrectly inserted ribonucleotide.
Describe the steps of termination in transcription.
Transcription proceeds until after the RNA polymerase transcribes a termination sequence in the DNA. This triggers the release of the RNA chain and the dissociation of the RNA polymerase from the DNA.
ο The transcribed terminator β an RNA sequence β codes for a polyadenylation signal sequence (AAUAAA)
ο In eukaryotic cell, the RNA polymerase continues transcription until at a point about 10 to 35 nucleotides downstream of the polyadenylation signal sequence. Proteins bind at this point to cut and free the pre-mRNA from the RNA polymerase.
ο This cleavage site on the mRNA is also the site of addition of poly (A) tail
Describe the steps in post-transcriptional modification.
- Addition of 5β cap consisting of methylated guanine (G) nucleotide / methylguanosine triphosphate. The cap: protects mRNA from degradation by hydrolytic enzymes, defines the 5β end of mRNA which recruits the small subunit of the ribosome for translation initiation, distinguishes mRNAs from other RNAs.
- RNA splicing: introns are removed while exons are spliced to form mature RNA, requiring ATP, carried out by spliceosomes.
- Addition of 3β Poly A tail: 200 adenine nucleotides, catalysed by poly A polymerase. The tail: protects mRNA from degradation by nucleases, increasing stability + facilitate export of mRNA out of the nucleus.
What does it mean by the genetic code is degenerate? And how does wobble base phenomenon occur as a result of this?
Degenerate but unambiguous: A single amino acid can be coded by more than one different codon. Only methionine (AUG) and tryptophan (UGG) are coded for by a single codon. But every codon codes for just one amino acid and thus the code is unambiguous. Most of the amino acids are encoded by degenerate codons that differ in the 3rd position β silent mutations. A single tRNA can recognise 2 or more of the degenerate codons. The violation of the usual rules of base pairing at the 3rd nucleotide of a codon is called wobble. Base-pairing at the 3rd base is not so specific, ie a change in the 3rd base by a mutation may still permit the correct incorporation of a given amino acid.
Define translation.
The process in which a polypeptide chain is synthesised by ribosomes using genetic information encoded in a mature mRNA template
Describe the structure and role of tRNA.
The secondary structure takes the form of a 2D cloverleaf, held by complementary base pairing within the single-stranded molecule.
ο On the anticodon loop, 3 unpaired bases form an anticodon.
ο· The tertiary structure is the result of actual twisting and folding of the secondary structure into
a compact 3D L-shaped structure maintained by hydrogen bonds.
The 3β end (CCA stem) of a tRNA molecule is the attachment site for a specific amino acid.
ο· There are around 45 different tRNAs in a typical eukaryotic cell, thus each amino acid can be carried by more than 1 type of tRNA.
ο· The 3D structure of tRNA is recognised by the enzyme aminoacyl-tRNA synthetase that catalyses the formation of an ester linkage (covalent bond) between the CCA stem and the specific amino acid.
tRNA serves as an adaptor molecule in the translation of an mRNA nucleotide sequence into the amino acid sequence + bring in specific amino acids corresponding to the sequence of codons in mRNA.
What is the role of rRNA?
- rRNA forms the core of the ribosome β it is the main constituent of the A and P sites and of the interface between the large and small ribosomal subunits.
- rRNA in the large ribosomal subunit has peptidyl transferase activity. It catalyses the formation
of peptide bonds between amino acids.
Describe the structure and role of ribosomes.
The small subunit (40S) contains an mRNA binding site, where the mRNA binds. The mRNA
binding site is associated along the surface close to the junction of the subunits. The large subunit (60S) has 3 binding sites for tRNA: A site, holds the incoming tRNA carrying the next amino acid, P site, holds the tRNA carrying the growing polypeptide chain, E site, site of release of the deacylated tRNA
Ribosomes are the organelles where synthesis of polypeptides
under the direction of mRNA occurs.
1. In protein synthesis, the ribosome provides an environment for specific recognition between
a codon of mRNA and an anticodon of tRNA.
2. The ribosome holds the tRNA and mRNA in close proximity. The ribosome positions the new amino acid for addition to the growing polypeptide.
3. rRNA in the large ribosomal subunit has peptidyl transferase activity. It catalyses the
formation of peptide bonds between amino acids
Translation is in the 5β to 3β direction.
Describe the steps in initiation of translation.
- ο· Eukaryotic initiation factors (eIFs) bind to the small subunit of a ribosome and position the
initiator tRNA, (tRNAimet) which carries a methionine to its P site, requiring GTP.
2.ο· The small subunit binds to the mRNA by recognition of its 5β cap.
ο· The small ribosomal subunit then moves downstream in the 5β to 3β direction along the mRNA in search of the start codon AUG - ο· The anticodon on initiator tRNA associates with the start codon on mRNA through complementary base pairing.
ο· Methionine is always the first amino acid in a newly formed polypeptide. The initiator tRNA has a unique anti-codon loop that is distinct from that of the tRNA that normally carries methionine.
ο· This is followed by the dissociation of eIFs (with the hydrolysis of GTP), which allows for the binding of the large ribosomal subunit, completing a eukaryotic 80S translation initiation complex.
ο· The initiator tRNA sits in the P site of the ribosome, and the initial methionine forms the Nβ terminus (βbeginningβ) of the polypeptide.
ο· The A site is vacant, waiting for entry of the next aminoacyl-tRNA complementary to the second codon of the mRNA.
Describe the steps in elongation of translation.
- ο· After the initiation complex has formed, an aminoacyl-tRNA carrying the 2nd amino acid in the chain binds to the ribosomal A site via the complementary base pairing between its anticodon and the codon in the mRNA exposed at the A site. The codon-anticodon interaction is held in place by hydrogen bonds.
ο· tRNAs are brought in by elongation factors. Energy is expended with the hydrolysis of GTP. - ο· When the second tRNA is bound to the ribosome, its amino acid is placed directly adjacent to the initial methionine
ο Peptidyl transferase in the large ribosomal subunit catalyses the formation of a peptide bond between the carboxyl end of methionine and the amino group of the 2nd amino acid.
ο The methionine is thus transferred to the 2nd amino acid carried by the aminoacyl-tRNA at the A site.
ο The ester bond between the initial methionine and the tRNA is broken to release the initial methionine (in the P site).
ο The deacylated tRNA lies in the P site, while the new peptidyl-tRNA has been created in the A site. - The ribosome is translocated one codon at a time in the 5β to 3β direction, guided by elongation factors, with the hydrolysis of GTP to provide energy. This relocates the initial deacylated tRNA (from the P site) to the E site from where it diffuses out of the ribosome, repositions the peptidyl-tRNA (at this point containing two amino acids) at the P site (from the A site), and exposes the next codon on the mRNA at the A site.
Describe the steps in termination in translation.
ο· Termination occurs when a stop codon (UAG, UAA, UGA) in the mRNA reaches the A site of the ribosome.
ο· A release factor binds directly to the stop codon in the A site, causing the addition of a water molecule instead of an amino acid to the polypeptide chain. This reaction frees the carboxyl end of the completed polypeptide from the tRNA in the P site by hydrolysis.
ο The polypeptide is released through the exit tunnel of the ribosomal large subunit.
ο· The ribosome then releases the mRNA and separates into the large and small subunits.
ο· tRNA molecules may then be recycled and used to form new aminoacyl-tRNA.
What are polyribosomes/polysomes and their advantage?
Ribosomes are often seen to occur in clusters known as polyribosomes or polysomes. When ribosomes occur as such aggregates, they are simultaneously translating polypeptides from the same mRNA strand.
As soon as the preceding ribosome has translated a portion of the nucleotide sequence, the 5β end of the mRNA is associated with a new ribosome. Each ribosome in the polysome independently synthesises a single polypeptide during its translation of the mRNA sequence.
Advantage: These multiple initiations (of translation) mean that many more polypeptide molecules can be made in a given time than would be possible if each had to be completed before the synthesis of the next could start.
Describe how polypeptides may be modified after translation.
- Attaching to it biochemical functional groups, such as acetate, methyl, phosphate and various lipids and carbohydrates.
ο· Glycosylation - the addition of specific short-chain carbohydrate / oligosaccharide is very common in membrane proteins.
ο· Reversible phosphorylation of threonine, serine, or tyrosine residues by kinases (which add a phosphate) and phosphatases (which remove the phosphate) plays an important role in the signal transduction processes regulating growth and cell cycle control. Phosphorylation may occur sequentially from one protein to another, resulting in a series of activations called a βphosphorylation cascadeβ. - Making structural changes, like the formation of disulfide linkages.
- Removing a sequence of amino acids from the protein, or cutting the peptide chain in the middle.
eg insulin is cut twice after disulfide bonds are formed, and a connecting peptide is removed from the middle of the chain. Proteolytic cleavage - The resulting protein consists of two polypeptide chains connected by disulfide bonds. - Attaching to it ubiquitin. Ubiquitin marks proteins for proteolysis by the proteasome. At least four ubiquitins are required on the substrate before a proteasome can bind to it. The polyubiquitin chain on a target protein is recognised by a specific receptor in the proteasome. In this manner, the selective degradation of proteins allows for the control of the length of time in which a protein can function.
