Eukaryotic Gene Expression Flashcards
define gene expression / the central dogma?
the process in which the information within a gene is used to synthesise, first RNA (in transcription) then a polypeptide (in translation) eventually to express the phenotype of an organism
what are the similarities between RNA and DNA? MEMORISE
- both polynucleotides
- sugar-phosphate backbone joined by phosphodiester bonds
- 3 bases: AGC
- complementary base pairing
- formed via condensation reaction
compare the differences between RNA and DNA MEMORISE
- molecular mass: RNA smaller (20-2000 kDa), DNA larger *100-150000 kDa)
- RNA is single-stranded, DNA is double-stranded
- monomers: RNA’s is ribonucleotides, DNA’s is deoxyribonucleotides
- chemical stability: RNA is less stable with additional reactive 2’ OH group, DNA lacks it and is more stable
- Nitrogenous bases: RNA has uracil and DNA has thymine
- ratio of purines to pyrimidines: RNA not 1:1, DNA is 1:1
- basic form: RNA has multiple (eg. mRNA, tRNA, rRNA), DNA has one
- location: RNA throughout the cell, DNA in nucleus (and mitochondria and chloroplasts, endosymbiotic theory)
- amount per cell: RNA varies, DNA constant
what are the roles of the different types of RNA (mRNA, tRNA, rRNA, snRNA, si/miRNA) MEMORISE
mRNA: carries information which codes for pp chains from DNA to ribosomes (only coding RNA)
tRNA: adaptor molecule in protein synthesis, translates mRNA codon sequence into aa sequence
rRNA: catalytic and structural roles in ribosomes
snRNA: catalytic and structural roles in spliceosomes
si/miRNA: 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 gene
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
a unit of inheritance located at a locus on the chromosome, which specifies a particular character of an organism
state the three key components of a eukaryotic gene
promotor, coding region, termination sequence
what is the role of the promoter in a eukaryotic gene?
contains the TATA box and transcription state site (+1: the nucleotide where RNA synthesis begins
- TATA box serves as binding site for general transcription factor (TFIID), which facilitates binding of RNA polymerase
- TATA box typically located 25 base pairs upstream (closer to 3’ direction)
- promoter determines which DNA strand used as template, not transcribed except for transcription start site
what is the role of the coding region in a eukaryotic gene?
- segment of DNA transcribed into single-stranded RNA molecule (pre-mRNA)
- bounded by transcription start and termination site
- only 1 strand serves as transcription template, read in 3’ to 5’ direction to facilitate RNA synthesis in 5’ to 3’ direction
- RNA synthesis occurs within transcription bubble, DNA transiently separated into single strands
what is the role of the termination sequence in a eukaryotic gene?
codes for polyadenylation signal sequence (AAUAAA) in the pre-mRNA
whole termination sequence is transcribed
- transcription termination (no more phosphodiester bonds formed)
describe the structure of RNA polymerase during transcription
RNA polymerase is an enzyme comprising of several protein subunits, found in the nucleoplasm
- responsible for RNA synthesis using ribonucleoside triphosphate (RTP) as its substrate
what are general transcription factors, and what is its role in transcription?
protein required for RNA polymerase molecule to bind to promoter and initiate transcription
help position RNA polymerase correctly at promotor and release RNA polymerase from promoter
what happens during the initiation stage of transcription?
general transcription factors (TFIID) binds to TATA box within promoter,mediating the binding of RNA polymerase to the promoter and forming a transcription initiation complex
RNA polymerase binding causes DNA double helix to unwind and separate both strands of DNA, breaking hydrogen bonds between complementary base pairs and creating a transcription bubble
one of the strands acts as a template for complementary base pairing to direct the assembly of incoming ribonucleotides, and RNA polymerase catalyses the formation of the first phosphodiester bond (marking the end of transcription initiation)
what happens during the elongation stage of transcription?
RNA polymerase moves along template DNA in 3’ to 5’ direction (reading it), transiently unwinding DNA double helix
ribonucleotides form complementary base pairs with DNA template, phosphodiester bond formed between free 5’ phosphate group of incoming nucleotide and free 3’ hydroxyl (OH) group of growing pre-mRNA chain
mRNA synthesised in 5’ to 3’ direction
RNA polymerase reanneals unwound DNA, and proofreads it
what happens during the termination stage of transcription?
RNA polymerase transcribes a termination sequence (polyadenylation signal sequence, AAUAAA) in DNA, triggering release of RNA chain and dissociation of RNA polymerase from DNA
in eukaryotic cells, RNA polymerase continues transcription until a point about 10-35 nucleotides downstream of polyadenylation signal sequence
cleavage site of mRNA is also the site of addition of poly (A) tail
what post-transcriptional modifications does pre-mRNA have to undergo?
- addition of 5’ methylguanosine cap
- RNA splicing
- addition of 3’ poly (A) tail
how and why does pre-mRNA undergo the addition of a 5’-methylguanosine cap?
5’ end of new RNA has a methylated guanine nucleotide / methylguanosine triphosphate “cap” added
protects mRNA from enzymatic degradation
defines the 5’ end of mRNA, serves to recruit the small subunit of the ribosome from translation initiation
distinguishes mRNAs from other types of RNA molecules
how and why does pre-mRNA undergo RNA splicing?
both exons (protein-coding sequences) and introns (non-coding sequences) are transcribed into pre-mRNA
RNA splicing removes introns and splices / joins remaining exons together to form mature mRNA, via the hydrolysis of ATP for energy
splicing is carried out by spliceosomes, a large complex with several subunits known as small nuclear ribonucleoproteins (snRNPs), each with small nuclear RNAs and a set of proteins
how and why does pre-mRNA undergo the addition of a 3’ poly (A) tail?
3’ end of pre-mRNA modified by addition of a series of approximately 200 adenine (A) nucleotides, referred to as the poly (A) tail, catalysed by the enzyme poly (A) - polymerase
protects mRNA from degradation, more stable
required to facilitate export of mRNA out of nucleus via nuclear pores
what are the general features of the genetic code (how does it work)?
the set of rules by which the DNA base sequence of a gene, through the intermediary mRNA codon sequence, is translated into the amino acid sequence of a pp
a list of the codons of mRNA, the sequence of triplet bases in non-template / non-transcribed strand of DNA, each codon is written as it appears in mRNA and reads in 5’ to 3’ direction
4^3 = 64 possible codons, three nucleotides per codon and four possible bases for each nucleotide position
of 64 possible codons, 61 code for the twenty possible amino acids and include a start codon, and three termination signals / stop codons
what are the key features of the genetic code (TUND)?
triplet: three nucleotide bases for one aa
universal: used by (almost) all organisms
non-overlapping: read continuously in 5’ to 3’ direction as successive groups of 3 nucleotides, one codon at a time without skipping, 3 possible reading frames for every mRNA sequence
degenerate: a single aa can be coded by multiple codon, only methionine (AUG) and tryptophan (UGG) coded for by a single codon each
but one codon = one aa (unambiguous)
silent mutations: most aa encoded by degenerate codons that differ in the 3rd position, mutations in this position can not be expressed, leading to wobble base phenomenon (base-pairing at third bp is not so specific, since tRNA can still recognise even if it’s wrong)
what does the start codon and stop codons define?
start signal is AUG, codes for incorporation of methionine, defining the first aa of pp chain and reading frame used from that point on
stop signals (UAA, UAG, UGA) has no tRNA with anticodon complementary to it
define translation
the process in which a polypeptide chain is synthesised by ribosomes using genetic information encoded in a mature mRNA template
state all the components of translation machinery
mature messenger RNA
transfer RNA
amino acids
aminoacyl-tRNA synthetase
ribosomal RNA and ribosome
translation factors
describe the structure and role of mature mRNA in translation
single RNA strand that exists for a relatively short time, obtained after pre-mRNA undergoes post-transcriptional modification
contains two regions:
1. protein-coding region consisting of series of codons representing aa sequence of pp, starting with the start codon AUG and ending with stop codon
untranslated regions (UTRs): additional sequence at 5’ end, preceding start codon (leader sequence or 5’ UTR) and following the stop codon (trailer sequence or 3’ UTR)
role: intermediate that carries a copy of DNA sequence information, each codon represents an aa, template for translation
describe the structure of tRNA used in translation MEMORISE
- small, ~80 nucleotides
- 2D cloverleaf with three loops held by complementary BP within single-stranded molecule (on anticodon loop, 3 unpaired bases form an anticodon, which binds to specific mRNA codon (secondary)
- 3’ end (CCA stem) of tRNA molecule is attachment site for a specific aa, around 45 different tRNAs in typical eukaryotic cell, each aa can be carried by >1 type of tRNA
- 3D structure of tRNA is recognised by enzyme aminoacyl-tRNA synthetase that catalyses formation of ester linkage (covalent bond) between CCA stem and specific aa
what is the role of tRNA in translation? MEMORISE
tRNA is an adaptor molecule, used to bring in specific aa
it can act as an adaptor because the anticodon can determine the specific aa attached to CCA stem and can form complementary base pairs with mRNA codon
how are amino acids attached to tRNAs?
enzyme: aminoacyl-tRNA synthetases
each aa is covalently attached to 3’ CCA stem via !ester linkage!, forming aminoacyl-tRNA, via hydrolysis of ATP
- active site of each aminoacyl-tRNA synthetase must be complementary to 3D conformation of specific aa and specific anticodon sequence of tRNA in order for them to bind
cells produce 20 different synthetase enzymes, one for each of the 20 distinct aa, each which recognise the specific anticodon on a tRNA as well as a specific aa
how are ribosomal subunits made?
- rRNA genes transcribed
- rRNA is processed, enters nucleus through pores
- assembled with proteins imported from cytoplasm in the nucleolus to make completed ribosomal subunits
- subunits exported via nuclear pores to cytoplasm
describe the structure and role of ribosomes
large ribonucleoprotein complex made of ribosomal proteins and ribosome RNA
prokaryotic ribosomes are ~70S, eukaryotic ribosomes ~80S
small subunit (40S) has mRNA binding site, large subunit (60S) has three tRNA binding sites:
A site (aminoacyl-tRNA site) holds incoming tRNA carrying the next aa
P site (peptidyl-tRNA site) holds the tRNA carrying the growing pp chain
E site (exit site) is the site of release of the deacylated tRNA
site of pp synthesis, provides environment for specific recognition between codon of mRNA and anticodon of tRNA, holds tRNA and mRNA in close proximity, positions the new aa for addition to growing pp, rRNA in large subunit has peptidyl transferase activity, catalyses formation of peptide bonds between aa
translated and read from 5’ to 3’
what are the three stages of translation, and the translation factors involved in each?
initiation (initiation factors), elongation (elongation factors), termination (release factors)
their respective factors use GTP as an energy source instead of ATP
what occurs during the initiation stage of translation?
brings together mRNA, initiator tRNA (met) and two ribosomal subunits, involving reactions preceding formation of first peptide bond
eukaryotic initiation factors (eIFs) bind to small ribosomal subunit, position initiator tRNA (tRNA carrying methionine) to P site, using GTP
small subunit binds to mRNA by recognition of 5’-methylguanosine cap
small ribosomal subunit moves downstream from 5’ to 3’ along mRNA in search of start codon AUG
anticodon on initiator tRNA (with met) binds to start codon on mRNA through complementary BP
dissociation of eIFs (with GTP hydrolysis) allows for binding of large subunit, making a eukaryotic 80S translation initiation complex
initiator tRNA sits in P site, initial methionine forms N-terminus of pp
A site of vacant, waiting for next aminoacyl-tRNA complementary to second mRNA codon
what occurs during the elongation and translocation stage of translation?
aminoacyl-tRNA carrying next aa binds to A site via complementary BP between its anticodon and codon in mRNA exposed at A site, held in place by hydrogen bonds
tRNA brought in by elongation factors, with the hydrolysis of GTP
when second tRNA bound to ribosome, its aa is directly adjacent to initial methionine, and peptidyl transferase in large subunit catalyses formation of a peptide bond between carboxyl end of previous aa and amino group of next aa, ester bond between previous aa broken to release it from P site, and deacylated tRNA (without aa) lies in P site, and new peptidyl-tRNA has been created in A site
translocation: ribosome moved down by one codon / three nucleotides in 5’ to 3’ direction with the hydrolysis of GTP, relocating initial deacylated tRNA from P to E site where it leaves, repositions peptidyl tRNA from A to P site, exposing the next codon at A site
what occurs during the termination stage of translation?
stop codon reaches A site of ribosome (UAG, UAA, UGA)
protein called release factor binds directly to stop codon in A site, causes addition of a water molecule instead of aa, freeing carboxyl end of completed pp from tRNA in P site by hydrolysis
what are polyribosomes / polysomes, and what are their advantages?
clusters / aggregates of ribosomes, simultaneously translating pp from the same mRNA strand, independently synthesising a single pp, more pp molecules made in a given time
where are free ribosomes found, and what proteins do they synthesise?
suspended in cytoplasm, synthesis proteins that dissolve in and exert effects in cytosol
where are bound ribosomes found, and what proteins do they synthesise?
attached to cytoplasmic side of endoplasmic reticulum
synthesise proteins of the nuclear envelope, ER, Golgi apparatus, lysosomes, vacuoles, plasma membrane, and secreted from cell
what are the stop codons?
UAA, UGA, UAG
u are annoying, you go away, you are gone
what is the role of a signal-recognition particle (SRP)?
shows that pp is to be secreted or embedded
(sequence of about 20 aa at or near the N-terminal of the pp, recognised as it emerges from ribosome by protein-RNA complex called a signal-recognition particle
functions as adaptor, brings ribosome to receptor protein built into ER membrane, growing pp snakes across ER membrane into cisternal space via protein pore)
what post-translational modifications can polypeptides undergo, and why?
polypeptide chains must coil and fold, assume specific 3D conformation to become functional protein
- attaching it to biochemical functional groups: glycosylation (to make glycoproteins) and reversible phosphorylation (signal transduction)
- making structural changes: formation of disulfide linkages
- removing a sequence of aa, cutting peptide chain: peptide hormone insulin cut twice, connecting peptide removed (proteolytic cleavage)
- attaching it to ubiquitin: marking proteins for proteolysis by proteasome (to break down peptide, selective degradation)
what is the role of RNA polymerase in transcription?
initiation:
- disrupts hydrogen bonds between complementary base pairs
- causes double helix to unwind and two strands to separate
elongation:
- reads DNA template strand in 3’ to 5’ direction
- catalyses the assembly of ribonucleotides which form complementary base pairs with the template
- forms phosphodiester bonds between free 5’ phosphate group of incoming ribonucleotide and free 3’ hydroxyl (OH) group of growing RNA polynucleotide chain (RNA synthesised in 5’ to 3’ direction)
- reanneals unwound DNA behind it
- proofreading functions
what is the role of rRNA?
- rRNA forms core of ribosome
- main constituent of A and P sites and the interface between large and small ribosomal subunits
- peptidyl transferase activity, catalyses formation of peptide bonds between aa
what is the role of rRNA? MEMORISE
- forms core of ribosome, main constituent of A and P sites and of interface btw large and small ribosomal subunits
- in large subunit has peptidyl transferase activity (catalyses formation of peptide bonds)
