eukaryotic gene expression

Question 1

what is the definition of gene expression?

Answer 1

gene expression is the process by which the information within a gene is used, first to synthesise mRNA through transcription and then to synthesise a polypeptide through translation, eventually to affect the phenotype of an organism. this is also referred to as the central dogma of molecular biology

Question 2

what is the definition of transcription?

Answer 2

it is 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, and is the first part of gene expression

Question 3

what is the definition of translation?

Answer 3

it is the process by which mRNA is used as a template for synthesis of polypeptides in the cytoplasm. the mRNA codon sequence is translated into the amino acid sequence of a polypeptide chain. 1 codon of amino acids specify 1 amino acid in a polypeptide chain, using a universal genetic code

Question 4

what are the similarities between RNA and DNA

Answer 4

1. both RNA and DNA are polynucleotides, where the components of each nucleotide monomer are: phosphate group, pentose sugar, and a nitrogenous base
2. both a strand of RNA and DNA has a sugar-phosphate backbone joined by phosphodiester bonds
3. both RNA and DNA make use of 3 nitrogenous bases: A,C and G
4. both RNA and DNA sequences are determined by complementary base pairing of nucleotide with a template
5. both RNA and DNA polynucleotide chains are formed via condensation reaction in which a water molecule is removed

Question 5

what are the differences between RNA and DNA

Answer 5

1. RNA is almost always a single-stranded helical molecule, which can be folded into a complex tertiary structure, while DNA is always a double-stranded helical molecule
2. RNA contains ribonucleotides as monomers, while DNA contains deoxyribonucleotides as monomers
3. RNA is chemically less stable, as ribose has an additional reactive 2’ OH group, while DNA is chemically more stable, as deoxyribose lacks the 2’ OH group which is replaced by a single H atom
4. RNA has a U nucleotide, while DNa has T nucleotide, which contains an additional 5’ methyl group as compared to U
5. RNA is synthesised in the nucleus but is found throughout the cell, while DNA is found almost exclusively in the nucleus with the exception of mitochondria and chloroplasts
6. the amount of RNA varies from cell to cell according to metabolic activity, while the amount of DNA is constant for all somatic cells of a species

Question 6

what are the functions of rRNA?

Answer 6

rRNA is the main constituent of the interface between the large and small subunits of the ribosome, allowing the mRNA to bind to the small ribosomal subunit through complementary base pairing.

Question 7

what is the definition of a gene?

Answer 7

a gene is 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. it is a unit of inheritance located in a locus on the chromosome, which specifics a phenotype of an organism

Question 8

what is the structure of the promoter?

Answer 8

the promoter contains the TATA box and the transcription start site, which is the nucleotide where RNA synthesis begins

Question 9

what is the function of the promoter?

Answer 9

the TATA box, which is typically located 25 base pairs upstream of the TSS, serves as a binding site for a general transcription factor. general transcription factors facilitates binding of RNA polymerase

the promoter determines which of the two strands of DNA is used as the template, and contains a short nucleotide sequence that is not transcribed except for the TSS

Question 10

what is the structure and function of the coding region?

Answer 10

the coding region is the segment of DNA that is transcribed into a single-stranded RNA molecule, and is bound by the TSS and termination site. only 1 of the 2 strands serves as the template for transcription, and is read in the 3’ to 5’ direction to facilitate synthesis of RNA in the 5’ to 3’ direction

Question 11

what is the structure and function of the termination sequence?

Answer 11

1. the termination sequence is found at the end of a gene and codes for a polyadenylation signal sequence (AAUAAA) in the pre-mRNA
2. the whole termination sequence is transcribed, resulting in transcription termination, where formation of phosphodiester bonds stops

Question 12

what is the structure of RNA polymerase?

Answer 12

RNA polymerase is an enzyme comprising of several protein subunits and is found in the nucleoplasm. the active site of RNA polymerase is complementary to that of the nucleotides on the template DNA strand

Question 13

what is the function of RNA polymerase?

Answer 13

1. during transcription, RNA polymerase reads the DNA template in the 3’ to 5’ direction, catalysing the assembly of ribonucleotides, which form complementary base pairs with the template, and the formation of phosphodiester bonds between the free 5’-phosphate group of the incoming NTP and the free 3’-OH group of the growing RNA chain
2. RNA is synthesised in the 5’ to 3’ direction, where RNA polymerase can simulataneously transcribe different parts of the same gene

Question 14

what is the function of general transcription factors?

Answer 14

general transcription factors are needed for RNA polymerase to bind to its promoter and initiate transcription, by:
1. positioning RNA polymerase correctly at the promoter
2. releasing RNA polymerase from the promoter to begin elongating the RNA against the DNA template once transcription has begun

Question 15

what are the three steps of initiation in transcription?

Answer 15

1. general transcription factors are assembled along the promoter, where they bind to the TATA box found within the promoter. general transcription factors mediate the binding of RNA polymerase to the promoter, forming a transcription initiation complex
2. binding of RNA polymerase to the promoter causes DNA double helix to unwind and separate. hydrogen bonds between complementary base pairs are disrupter, and a transcription bubble exposing a short stretch of nucleotides on each strand is created
3. the DNA template strand directs the assembly of incoming NTPs through complementary base pairing, where RNA polymerase form phosphodiester bonds

Question 16

what are the three steps of elongation in transcription?

Answer 16

1. as RNA polymerase moves along the template DNA in the 3’ to 5’ direction, DNA continues to transiently unwind
2. NTPs form complementary base pairs with the DNA template. as each NTP is brought in, its two terminal phosphates are removed, and the remaining free 5’-phosphate group is added to the free 3’-OH group of the growing RNA chain via the formation of a phosphodiester bond
3. RNA polymerase reanneals the unwound DNA, dissociating the growing RNA chain from the template. RNA polymerase also carries out proofreading functions and is responsible for the removal of any incorrect inserted NTPs

Question 17

what are the steps in termination in transcription?

Answer 17

1. upon transcription of the termination sequence AAUAAA, this triggers the release of the RNA chain and the dissociation of the RNA polymerase from the DNA.
2. 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

Question 18

what are the functions of the 5’ methylated guanine nucleotide cap on the end of a new RNA molecule?

Answer 18

1. the 5’ cap protects the mRNA from degradation by hydrolytic enzymes such as nucleases
2. the 5’ cap defines the 5’ end of the mRNA, which serves to recruit the small subunit of the ribosome for translation initiation
3. the 5’ cap distinguishes mRNAs from other types of RNA molecules

Question 19

what is the process of RNA splicing?

Answer 19

1. splicing is carried out by spliceosome which is a large complex comprising of several subunits known as small nuclear ribonucleoproteins (snRNPs). each snRNP contains small nuclear RNAs and a set of proteins
2. during this process, introns are removed while the remaining exons are spliced to form mature RNA, requiring the hydrolysis of ATP

Question 20

what is the process of the addition of 3’ poly(A)tail?

Answer 20

1. immediately after termination occurs, the 3’ end of the pre-mRNA is modified by addition of a series of approximately 200 adenine molecules, referred to as the poly(A)tail.
2. this process is catalysed by the enzyme poly(A)-polymerase

Question 21

what are the functions of the 3’ poly(A)tail?

Answer 21

1. the 3’ poly(A)tail protects the mRNA from degradation by nucleases, making the mRNA a more stable template for translation in the cytoplasm
2. the 3’ poly(A) tail is required to facilitate export of mRNA out of the nucleus via nuclear pores

Question 22

what are the key features of the genetic code?

Answer 22

1. the genetic code is a triplet code - each mRNA codon that specifies an amino acid in a polypeptide chain consists of three nucleotide bases
2. the genetic code is almost universal - the same code is used by almost all living organisms
3. the genetic code is continuous and non-overlapping - the nucleotides in the mRNA are read continuously as successive groups of 3 nucleotides, one codon at a time without skipping any nucleotides.
4. the genetic code is degenerate but unambiguous - a single amino acid can be coded by more than one different codon. however, 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 of the codon, and are termed as silent mutations
5. wobble base phenomenon occurs - in some cases, a single tRNA can recognise 2 or more of the degenerate codons, and this violation of the usual rules of base pairing at the 3rd nucleotide of a codon is called wobble, suggesting that the base pairing at the 3rd base is not so specific
6. the genetic code has start and stop codons, where the start codon is AUG which codes for the incorporation of methionine, while the stop codons are UAA, UAG and UGA, which do not code for any amino acid

Question 23

what is the structure of mature mRNA?

Answer 23

mature mRNA is obtained after the pre-mRNA undergoes post-transcriptional modification. a mature mRNA molecules contains:
1. protein-coding region, which consists of a series of codons representing the amino acid sequence of the polypeptide, starting with the start codon AUG and ending with a stop codon UAA/UAG/UGA
2. untranslated regions, which is an additional sequence at the 5’ end, preceding at the start codon, known as the leader codon, and following the stop codon known as the trailer sequence

Question 24

what is the function of mature mRNA?

Answer 24

1. mature mRNA serves as an intermediate that carries the copy of DNA sequence information that encodes proteins. each codon within the coding region of the mRNA represents an amino acid in the corresponding amino acid sequence in the protein
2. mature mRNA acts as a template for translation and guides the assembly of amino acids into a polypeptide chain

Question 25

what is the structure of tRNA?

Answer 25

1. the secondary structure of tRNA takes the form of a 2D clover leaf, held by complementary base pairing within the SS molecule. there are three loops, and on the anticodon loop, 3 unpaired bases form an anticodon, which binds to a specific mRNA codon via complementary base pairing 2. 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 3. the 3’ end of a tRNA molecule in the is the attachment site for a specific amino acid. there are around 45 different tRNAs in a typical eukaryotic cell 4. the 3D structure of tRNA is recognised by the enzyme aminoacyl-tRNA synthetase that catalyses the formation of an ester linkage between the CCA stem and the specific amino acid

Question 26

what are the functions of tRNA?

Answer 26

tRNA serves as an adaptor molecule in the translation of a mRNA nucleotide sequence into the amino acid sequence in a polypeptide, because of: 1. the ability of the anticodon to determine the specific amino acid attached to the CCA stem 2. the ability of the anticodon to form complementary base pairs with the mRNA codon

Question 27

how are amino acids activated by aminoacyl-tRNA synthetase?

Answer 27

1. cells produce 20 different synthetase enzymes, once for each of the 20 distinct amino acids. each of the 20 different synthetase enzymes must recognise the specific anticodon on a tRNA as well as a specific amino acid 2. each of the 20 different synthetase enzyme covalently attaches a specific amino acid to the 3’ CCA stem of its appropriate set of tRNA molecules via an ester linkage, forming aminoacyl-tRNA, requiring the hydrolysis of ATP 3. the active site of each aminoacyl-tRNA synthetase must be complementary to the 3D conformation of the specific amino acid and specific anticodon sequence of the tRNA in order for binding to occur 4. the resulting aminoacyl-tRNA is released from the synthetase enzyme and delivers its amino acid to a growing polypeptide chain on a ribosome

Question 28

what is the structure of rRNA?

Answer 28

1. rRNA constitutes approximately 80% of RNA in rapidly dividing cells 2. rRNA is processed and assembled with proteins imported from the cytoplasm in the nucleolus. completed ribosomal subunits are then exported via nuclear pores to the cytoplasm 3. 18S, 5,8S and 28S rRNA are made by chemically modifying and cleaving a single large precursor rRNA. these chemical modifications are made at a specific position in the precursor rRNA, and aid in the folding and assembly of the final rRNA, subtly altering the function of ribosomes 4. 5S rRNA is synthesised from a separate cluster of genes and do not require chemical modification

Question 29

what is the function of rRNA/

Answer 29

1. rRNA forms the core of the ribosome, and is the main constituent of the A and P sites and of the interface between the large and small ribosomal subunits 2. rRNA in the large ribosomal subunit has peptidyl transferase activity, and catalyses the formation of peptide bonds between amino acids

Question 30

what is the structure of ribosomes?

Answer 30

the ribosome is a large ribonucleoprotein complex composed of ribosomal proteins and rRNA. the small subunit (40S) contains an mRNA binding site, which is associated along the surface close to the junction of the subunits. the large subunit (60S) has an A, P and E binding sites for tRNA. the aminoacyl-tRNA holds the incoming tRNA carrying the next amino acid to be added. the peptidyl-tRNA site holds the tRNA carrying the growing polypeptide chain. the exit site is the site of release of the deacylated tRNA

Question 31

what is the function of ribosomes?

Answer 31

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, and in doing so the ribosome positions the new amino acid for addition to the growing polypeptide 3. the rRNA in the large ribosomal subunit has peptidyl transferase activity, and catalyses the formation of peptide bonds between amino acids

Question 32

what are the steps of initiation in translation?

Answer 32

1. eukaryotic initiation factors bind to the small subunit of a ribosome and position the initiator tRNA, which carries a methionine to its P site 2. this subunit binds to the mRNA by recognition of its 5’ cap, and the ribosomal subunit then moves downstream in the 5’ to 3’ direction along the mRNA in search of the start codon AUG, which signals the start site of translation 3. the anticodon on initiator tRNA associates with the start codon on mRNA through complementary base pairing, and this is followed by the dissociation of eukaryotic initiation factors, which allows for the binding of the large ribosomal; subunit, completing a eukaryotic 80s translation initiation complex

Question 33

what are the steps of elongation and translocation in translation?

Answer 33

1. 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 2. tRNAs are brought in by elongation factors, and energy is expended with the hydrolysis of GTP 3. 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. the methionine is thus transferred to the 2nd amino acid carried by aminoacyl-tRNA at the A site. the ester bond between the initial methionine and the tRNA is broken to release the initial methionine. 4. translocation relocates the initial deacylated tRNA from the P site to the E site from where it diffuses out of the ribosome. this repositions the peptidyl-tRNA at the P site from the A site and exposes the next codon an the mRNA at the A site

Question 34

what are the steps of termination in translation?

Answer 34

1. termination occurs when a stop codon in the mRNA reaches the A site of the ribosome. the base triplets UAG, UAA and UGA act as signals to stop translation 2. 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 frees the carboxyl end of the polypeptide from the tRNA in the P site by hydrolysis. the polypeptide is released through the exit tunnel of the ribosomal large subunit, and the translation complex comes apart 3. tRNA molecules may be recycled and used to form new aminoacyl-tRNA

Question 35

what are the advantages of polyribosomes?

Answer 35

these multiple initiations of translation mean that many more polypeptide molecule 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, as each ribosome in the polysome independently synthesises a single polypeptide during its translation of the mRNA sequence

Question 36

how are polypeptides of proteins destined for the endomembrane system or for secretion?

Answer 36

these polypeptides are marked by a signal peptide, which is a sequence of about 20 amino acids at or near the N-terminal of the polypeptide, and is recognised as it emerges from the ribosome by a protein-RNA complex called a signal-recognition particle. the SRP functions as an adaptor that brings the ribosome to a receptor protein built into the ER membrane. polypeptide synthesis continues there

Question 37

how is attaching biochemical functional groups to polypeptides carried out?

Answer 37

1. glycosylation is very common in membrane proteins, as these proteins known as glycoproteins are important in many cell processes, including signal recognition and activation of an immune response 2. reversible phosphorylation by kinases and phosphatases play an important role in the signal transduction processes regulating growth and cell cycle control

Question 38

how is removing a sequence of amino acids carried out?

Answer 38

for instance, insulin is cut twice after disulfide bonds are formed, and a connecting peptide is removed from the middle of the chain, known as proteolytic cleavage

Question 38

how is attaching upiquitin to polypeptides carried out?

Answer 38

upiquitin 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