Week 5 Flashcards
draw a simplified model of prokaryotic gene expression and of eukaryotic gene expression
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.
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
