W5+ W6 Flashcards
define a gene
the entire nucleic acid sequence (usually DNA) that is necessary for the synthesis of a protein (and its variants) or RNA. In other words, genes are segments of DNA that are transcribed into RNA
two types of genes when transcribed
- resulting RNA encodes a protein
- resulting RNA functions as RNA and may not be translated into protein
RNA nucleotides are added in which direction?
5’ to 3’ (reads template strand 3’ TO 5’)
one important difference between RNA polymerase and DNA polymerase
ribonucleoside triphosphate used (ATP, UTP, CTP, GTP)
coding strand
non-template strand - almost identical to the RNA strand formed
RNA nucleotides are linked by
phosphodiester bonds
DNA-RNA helix in RNA polymerase held together by
base pairing
template for RNA polymerase is what type of DNA?
ssDNA
label a functional RNAP
DNA/RNA duplex
short region of DNA/RNA helix
nascent RNA
newborn RNA that is being synthesised
promoter
signals to the RNA polymerase to start transcribing
transcription cycle steps
- Sigma factor binds to RNAP and finds promoter
sequence - Localized unwinding of DNA, a few short
RNAs synthesized initially & then RNAP clamps down–sigma factor released - NB: no primer needed
- Elongation
- Termination & release of RNA (terminator sequence is transcribed)
are sigma factors promoter-specific?
different sigma factors recognise different promoter sequences
function of sigma factor
a bacterial transcription initiation factor that enables specific binding of RNA polymerase (RNAP) to gene promoters - reads sequences and recognises where to bind (by taking asymmetry into account) and which strand to bind to.
how is the sequence numbered?
counting backward from the start site using negative numbers
upstream vs downstream
Upstream is toward the 5’ end of the synthesised RNA molecule, and downstream is toward the 3’ end.
promoter consensus sequences
-10 has the consensus sequence TATAAT. · The sequence at -35 has the consensus sequence TTGACA
RNA secondary structure
gerald!
- conventional base pairs are made among different parts of the molecule
- hairpins etc
terminator sequence
GC rich areas followed by AT rich areas on the template strand (results in AUUUUU)
- strong hydrogen bonding between GC rich areas on RNA strand forms a termination hairpin, helping pull the RNA away
difference between promoter and terminator sequences
promoter sequences not usually transcribed
key changes in efficiency in the transcription cycle
- initial steps of RNA synthesis are relatively inefficient
- this is different from the elongation mode of RNA polymerase, which is highly processive
how do termination signals help to dissociate the RNA transcript from the polymerase?
disrupt H-bonding of new mRNA
transcript with DNA template
difference between gene expression in prokaryotes and eukaryotes
translation in prokaryotes can occur concurrently with transcription due to the absence of the nucleus.
in eukaryotes, pre-mRNA is altered to become mature mRNA which is then exported out of the nucleus and translated
what are UTRs ?
untranslating regions: segments of an mRNA molecule that are not translated into protein. They are located at both the 5’ and 3’ ends of the mRNA.
what is the function of UTRs?
- regulation of translation: UTRs can influence the efficiency and rate at which a protein is synthesized by affecting ribosome binding and initiation.
- mRNA stability: UTRs play a role in determining the half-life of an mRNA molecule, thereby influencing how long it remains available for translation.
draw a simplified model of prokaryotic gene expression and of eukaryotic gene expression
mRNAs
messenger RNAs, code for proteins
rRNAs
ribosomal RNAs, form the basic structure of the ribosome and catalyse protein synthesis
tRNAs
transfer RNAs, central to protein synthesis as the adaptors between mRNA and amino acids
telomerase RNA
serves as the template for the telomerase enzyme that extends the ends of chromosomes
snRNAs
small nuclear RNAs, function in a variety of nuclear processes, including the splicing of pre-MRNA
snoRNAs
small nucleolar RNAs, help to process and chemically modify rRNAs
lncRNAs
long noncoding RNAs, not all of which appear to have a function; some serve as scaffolds and regulate diverse cell processes, including X-chromosome inactivation
miRNAs
microRNAs, regulate gene expression by blocking translation of specific mRNAs and causing their degradation
siRNAs
usually double stranded. small interfering RNAs, turn off gene expression by directing the degradation of selective mRNAs and helping to establish repressive chromatin structure
piRNAs
iwi-interacting RNAs, bind to kiwi proteins and protect the germ line from transposable elements
distinguish between RNAPs in prokaryotes and eukaryotes
in prokaryotes, there is only one RNAP
in eukaryotes, there are three: RNA polymerase I, II, and III
structure and function of RNAPs in eukaryotes
- each RNAP is a multi-subunit protein
- each RNAP is responsible for transcription of different RNAs
genes transcribed by RNA polymerase I
most rRNA genes
genes transcribed by RNA polymerase II
all protein-coding genes, miRNA genes, ;lus genes for other noncoding RNAs (eg those of the spliceosome)
genes transcribed by RNA polymerase III
tRNA genes, 5S rRNA gene, genes for many other small RNAs
distinguish between eukaryotic RNAP II and bacterial RNAP structure
- bacterial RNAP has 5 subunits, eukaryotic RNA Pol II has 12
- RNA pol II has a special carboxyl terminal domain (CTD) not found in bacterial or other eukaryotic RNAPs
why do eukaryotic RNA polymerases require transcription factors?
- these proteins help position them at the promoter
- fulfil a similar role to the sigma subunit of the bacterial RNA polymerases
- eukaryotic RNA polymerases need to deal with chromosomal structures so more/diverse transcription factors are needed as finding the gene to transcribe is harder
sigma subunits
responsible for determining the specificity of promoter DNA binding and efficient initiation of RNA synthesis
eukaryotic promoters
TATA box:
- helps position RNAP II
- A/T-rich sequence highly conserved
- found at ~30bp upstream from start site for transcription
- common, but there are also many other types of promoter sequences (elements)
steps in the initiation of transcription
- binding of TBP (TATA box binding protein) subunit of TFIID (Transcription factor II D) to TATA box promoter in the minor groove, bending and distorting the DNA which makes all components proximal.
- this mobilizes the binding of TFIIB complex adjacent to the TATA box
- other transcription factors bind, helping orient and bind RNAP II to the DNA at the TSS (transcription start site)
- the helicase activity of TFIIH uses ATP to pry apart DNA strands at the TSS
- TFIIH also phosphorylates the C-terminal domain of RNA polymerase II, activating it so that transcription can begin.
major vs minor groove of DNA
The major groove occurs where the backbones are far apart, the minor groove occurs where they are close together.
why are additional factors required for transcription elongation in eukaryotes?
elongation factors act like a wedge prying DNA off histone so RNAP can perform its function. this prevents RNAP from stalling. Proteins are also involved in then reassembling the nucleosome
describe the RNA polymerase II C-terminal domain
- carboxyl terminal domain on the largest subunits
- consists of tandem repeats of 7 amino acids
- this happens in RNAP II only
- repeat: (N terminal) Tyr-Ser-Pro-Thr-Ser-Pro-Ser (COOH terminal)
- Ser AAs are phosphorylated by TFIIH in different patterns
- Phosphorylation of the CTD serves as a binding platform for different RNA-processing factors, including those involved in capping, splicing, and polyadenylation.
how many repeats of AAs does yeast enzyme vs human enzyme have?
yeast - 26
human - 52
review qs on transcription initiation:
how is RNA polymerase II activated? phosphorylation
what is phosphorylated? See on CTD of RNAPII
how many proteins are involved in initiation eukaryotic transcription? >100 subunits of many proteins
3 main steps of mRNA processing
- addition of 5’ cap
- splicing - removal of introns
- processing and polyadenylation of 3’ tail
phosphorylation of C-terminal tail of RNAP II results in binding of:
- RNA processing proteins
- additional phosphorylation of CTD, including Ser 2 (done by other enzymes)
capping proteins are attracted by
Ser 5 phosphorylation
splicing proteins are attracted by
Ser 2 phosphorylation, which is not done by TFIIH but by a different kinds
5’ pre-mRNA capping
- requires 3 enzymes
- 5’ cap consists of 7-methylguanosine and a 5’-5’ triphosphate bridge, which is not recognised by exonucleases so can’t be degraded
- helps to protect RNA from nucleases
- completed before mRNA full transcribed
gene structure in prokaryotes vs eukaryotes
prokaryotes: promoter -> bacterial gene (coding sequence)
eukaryotes - promoter -> coding sequences (exons) -> noncoding sequences (introns) which are later spliced out ///
how are introns removed from pre-RNA?
- branch point A (an adenosine (A) residue within the intron, located 20–50 nucleotides upstream of the 3’ splice site) is recognized by splicing factors.
- the 2’-OH group of the branch point A attacks the 5’ splice site, forming a 2’-5’ phosphodiester bond.
This creates a looped structure called a lariat, where the intron is circularized with the branch point A at its center. - the 3’ OH of the upstream exon attacks the 3’ splice site, releasing the lariat intron and joining the exons together.
why is the catalytic mechanism of RNA splicing RNA dependent?
- the 2’ OH group of the ribose sugar is not present in deoxyribose, so the DNA doesn’t self-splice
how are snRNPs involved in the splicing reaction for most eukaryotic pre mRNAs?
- pre-mRNAs are not able to self-splice
- spliceosomes contain snRNAs bound to protein (snRNPs) plus other associated proteins
- spliceosomes assemble on mRNA to remove introns
- when splicing is complete, an exon junction complex is added
order in which snRNPs are used
- U1 snRNP binds to the 5’ end of the intron; U2 snRNP binds to the 3’ end of the intron
- U6 replaces U1 (a form of checking)
- active site of spliceosome is created by U2 and U6
- splicing occurs by a transesterification reaction
- exon junction complex is added between exon 1 and exon 2 to signal to cell that mRNA is properly spliced
what is a secondary function of alternative RNA splicing?
it increases the number of possible gene products
examples of abnormal splicing
- a single-nucleotide change that destroys a normal splice site, thereby causing exon skipping
- a single-nucleotide change that destroys a normal splice site, thereby activating a cryptic splice site
- a single-nucleotide change that creates a new splice site, thereby causing a new exon to be incorporated
??transcirption of the consensus sequences and recruitment of 3’ end modifying proteins
??
3’ end processing
- consensus sequences direct cleavage and polyadenylation of the 3’ end
- 3’ end processing proteins move from CTD to mRNA
- cleavage and addition of a poly-A 3’ tail along with poly-A binding proteins result in the mature mRNA
- CTD is dephosphorylated, causing RNAP to detach
how is mature mRNA exported from the nucleus to the cytosol?
leaves nucleus through nuclear pore complex in the nuclear envelope. proteins in the NPC check sequence and associated proteins. in the cytosol, proteins are exchanged with initiation factors for protein sequence
how does transcription differ from DNA replication in terms of the binding between the DNA and the strand being formed?
RNA strand does not remain hydrogen-bonded to the DNA template strand. Instead, just behind the region where the ribonucleotides are being added, the RNA chain is displaced and the DNA helix reforms.
two main differences between RNA and DNA polymerase
- RNAP turns ribonucleoside triphosphates as substrates not deoxyribocleoside triphosphates
- RNAP can start RNA chain without a primer and do not accurately proofread their work
why is the asymmetry of the promoter important?
it orients the polymerase and determines which DNA strand is transcribed
how does the sigma factor see the promoter within the double helix?
each base presents unique features to the outside of the double helix, allowing the sigma factor to initially identify the promoter sequence without having to separate the entwined DNA strands
four differences between eukaryotic and prokaryotic initiation of transcription
- eukaryotic uses more than one RNAP; RNAP I and II transcribe genes encoding tRNA, rRNA, and other RNAs that play structural and catalytic roles . RNAP II transcribes the rest (inc proteins)
- bacterial RNAP relies on a single accessory protein - sigma factor - to initiate transcription. Eukaryotic RNA polymerase requires many general transcription factors
- in eukaryotes, single genes are controlled by a large variety of regulatory DNA sequences; more complex forms of transcriptional regulation than bacteria
- eukaryotic transcription initiation must deal with the packing of DNA into nucleosomes and higher-order forms of chromatin structure
how are mRNA processing steps different in bacteria compared to eukaryotes?
in bacteria, the 5’ end of an mRNA molecule is simply the first nucleotide of the transcript and the 3’ end is simply the end of the chain synthesised by RNAP
how do RNA processing proteins know where to assemble?
phosphorylation of the tail of RNA polymerase II allows the proteins to assemble there
what are ribozymes
RNA molecules that have the ability to catalyze specific biochemical reactions, including RNA splicing in gene expression
how is the nucleus functionally organised?
RNAs are synthesised and processed within biomolecular condensates that serve to condensate these components:
- large nucleoli: subcompartments in which rRNAs are synthesised and ribosomes assembled
- Cajal bodies: maturation of snRNPs and snRNAs takes place
- interchromatin granule clusters: stockpiles of snRNPs and other RNA-processing components used in the production of mRNA
how are biomolecular condensates held together>
by the weak noncovalent interactions that continually form and break between their constituent macromolecules
how does the cell ensure that only mature eukaryotic mRNAs are exported from the nucleus?
- transport of mRNA from the nucleus to the cytosol is highly selective
- 5’ cap and poly-A tail of a mature mRNA molecule are marked by proteins that recognise these modifications
- successful splices are marked by exon junction complexes
- once an mRNA is deemed export ready a nuclear transport receptor associates with the mRNA and guides it through the nuclear pore and into the cytosol
why is the lifespan of an mRNA important?
- it helps the cell control how much protein will be produced
- this is usually controlled by the 3’ UTR
- each mRNA molecule is eventually degraded into nucleotides by ribonucleases (RNases) in the cytosol
3’ end processing
- consensus sequences direct cleavage and polyadenylation of the 3’ end
- 3’ end processing proteins move from CTD to mRNA
- cleavage and addition of a poly-A 3’ tail along with Poly A-binding proteins result in the mature mRNA
state 4 features of the genetic code
- universal for almost all genomes
- codons are read as mRNA triplets encoding all 20 amino acids
- redundancy: multiple codons for most amino acids
state the stop and start codons
AUG - methionine - start
UAA/UAG/UGA - stop codons (don’t code for an AA)
describe the possible mutations that could occur within a reading frame
- nucleotide-pair substitution: silent (the amino acid does not change)
- nucleotide-pair substitution: missense (the amino acid is changed into a different amino acid)
- nucleotide-pair substitution: nonsense (leads to a premature stop codon)
- 1 nucleotide-pair deletion (frameshift causing immediate missense - all following amino acids likely to be wrong)
- 1 nucleotide-pair insertion (frameshift causing immediate nonsense)
- 3 nucleotide-pair deletion (no frameshift, but amino acid changes)
why is the cloverleaf appearance of tRNA so important?
- tRNA acts as an adaptor molecule
- its secondary structure is critical to RNA function
- the anticodon loops allows the anticodon to base pair with the mRNA codon in a complementary and antiparallel way
draw a standard tRNA structure and label it
are there the same number of tRNAs as there are anti-codons?
if we take out the stop codons, we have 61 possible anti-codons, but bacteria have 31 tRNAs and humans have 48.
two possible strategies:
- more than 1 tRNA for many amino acids
- some tRNAs can recognise and base pair with more than one codon
describe the wobble position of a tRNA molecule
- third base pair between codons/anticodons
- flexible: base pair does not have to be perfect
- saves the number of tRNAs that have to be produced
- may ensure mutations have a lesser effect
wobble codon base and possible anticodon bases in bacteria
U = A, G, I
C = G, I
A = U, I
G = C, U
wobble codon base and possible anticodon bases in eukaryotes
U = A, G, I
C = G, I
A = U
G = C
what is I?
Inosine, which represents a post-transcriptional modification of adenosine
seen the presence of a wobble position, how fidelity in base pairing between codons and anticodons ensured?
- sequential steps in ensuring fidelity:
- aminoacyl-tRNA synthetases: check compatibility of amino acid and tRNA then makes a high-energy bond using ATP
- base pairing between mRNA and tRNA in ribosome - error correction by aminoacyl tRNA synthetase:
- by hydrolytic editing to break the high energy between the tRNA and amino acid
how is recognition of a specific tRNA by its synthetase achieved?
- identifying the tRNA anticodon nucleotides
- recognising the nucleotide sequence of the acceptor stem/arm
- reading nucleotide sequences at additional positions on the tRNA
label a diagram of tRNA synthetase binding to tRNA
location of ribosomes in a eukaryotic cell
- on endoplasmic reticulum
- in cytosol
location of ribosomes in a prokaryotic cell
- in cytosol
distinguish between the function of the large and small ribosomal subunit
The small subunit (40S in eukaryotes) decodes the genetic message and the large subunit (60S in eukaryotes) catalyzes peptide bond formation.
how is peptide synthesis made energetically favourable?
with the energy stored in covalent bond between the amino acid and the tRNA in P site
A, P, E site
A site - aminoacyl site - aminoacylated tRNA enters here
P site - peptide site - where the peptide bond is formed
E site - exit site
what catalyses the formation of the peptide bond?
peptidyl transferase activity of the rRNA in the large subunit - ensures the high energy bond between the amino acid and the tRNA provides the energy for forming the new peptide bond
which translocates first - the large or the small ribosomal subunit?
the large subunits translocates first; then, the small subunit
describe the structure of a prokaryotic ribosome (eukaryotic is very similar)
- ribosome is a ribozyme
- L1 protein is involved in folding and stabilising RNAs
- 5S RNA: component of the large ribosomal subunit thought to enhance protein synthesis by stabilization of a ribosome structure
- 23S rRNA: for tRNA binding in the P site of the large ribosomal subunit
elongation factors
- EF-Tu (pro) / EF1 (eu) checks aminoacyl tRNA.
- if base pairing is not correct, EF-Tu is not released and peptide bond can’t form.
- if base-pairing is correct, GTP is hydrolysed and EF-Tu is released
- there is also a slight delay before the formation of the peptide bond which allows one last check for accurate base pairing - EF-G (pro / EF2 (eu) helps the ribosome to move the mRNA forward one codon and helps speed up elongation of the polypeptide chain
- requires energy from hydrolysis of GTP-> GDP
- without this EF, translation is very slow
can ribosomes perform protein synthesis without the aid of elongation factors?
yes, but it is much slower, more inefficient, and less accurate
distinguish between mRNA structure in bacteria (prokaryotes) and eukaryotes
- Prokaryotic mRNA has a Shine-Dalgarno sequence for ribosome binding, while eukaryotic mRNA has a 5’ cap and poly-A tail for protection and ribosome binding.
- The first amino acid differs: formylmethionine in prokaryotes and methionine in eukaryotes.
- mRNA is polycistronic in prokaryotes (produces multiple proteins) and monocistronic in eukaryotes
initiation of translation in prokaryotes
- shine-dalgarno sequences on mRNA base pair with rRRNA in small ribosomal subunits
- positioning of small ribosomal subunits to initiating AUG codons on mRNA also requires Initiation Factors (IFs)
- fMethionine aminoacyl tRNA binds to initiator codon in the P-site of small ribosomal subunit. Formyl group has been covalently added to R group of Met.
- large ribosomal subunit binds
initiation of translation in eukaryotes
- The initiator tRNA, charged with methionine, and various translation initiation factors, bind tightly to the P site of the small ribosomal subunit.
- The small ribosomal subunit attaches to the 5’ end of an mRNA molecule and scans along the mRNA until it encounters a start codon (AUG).
- Upon finding this start codon, translation initiation factors dissociate and the large ribosomal subunit binds
- a charged tRNA binds to the second codon
- first peptide bond forms
function of initiation factors that bind at the 5’ cap and poly-A tail of the mRNA in eukaryotes
- these can bind to each other, bringing the 5’ end closer to the 3’ end, thus circularising RNA
- checks both ends to ensure modification has been correct and RNA is ready for translation
- facilitates ribosome recycling and enhances translation efficiency by making it easier for ribosomes to reinitiate translation on the same mRNA molecule
how is translation terminated?
- there is binding of a release factor to the A site, which breaks the bond between the tRNA and amino acid in the P site
- hydrolysis causes the polypeptide chain to be released and the ribosome dissociates
is the human translation release factor a protein or a tRNA?
eRF1 (in eukaryotes), RF1, and RF2 (in prokaryotes) are proteins
polyribosomes/polysomes
- arises from circularising RNA
- makes translation much more efficient
- occurs in both eukaryotes and prokaryotes
- multiple ribosomes attached to the same mRNA strand to produce multiple proteins from the same mRNA
- otherwise, protein synthesis is relatively slow
- ribosomes are spaced every 80nt
how is protein folding conducted?
- most proteins can fold on their own but this is usually incorrect and slow
- chaperone proteins - Hsp60 and Hsp70 - are used to help correctly fold the proteins
post-translational modifications
- many proteins require post-translational modifications such as phosphorylation and glycosylation (common in membrane proteins)
- covalent modifications may be required to make a protein active or recruit a protein to the correct membrane/organelle
when do proteins need to be degraded?
- haven’t folded properly
- amino acids have been modified
describe protein degredation
proteins targeted for degradation have a small protein called ubiquitin covalently attached to them, which directs them to the proteasome where they are degraded by proteases, and the amino acids recycled into new proteins by the cell
describe the structure of a proteasome
- polyubiquitin-binding site
- central cylinder (containing active sites of proteases)
- stopper
composition of a ribosome
two thirds RNA and one third protein by weight
main role of the ribosomal proteins
- to help fold and stabilise the RNA core, while permitting the changes in rRNA conformation the are necessary for this RNA to catalyse efficient protein synthesis
how is the catalytic site for peptide bond formation on ribosomes formed?
- by the 23S rRNA of the large subunit
- peptide transferase
- highly structured pocket that precisely orients the two reactants - polypeptide and incoming amino acid - increasing the likelihood of a productive reaction
how do most antibodies work?
act bye inhibiting bacterial, but not eukaryotic, gene expression.
