3.3 transcription - mechanism Flashcards
location of transcription and fate of RNA in bacteria vs eukaryotes
Bacteria
- occur in cytoplasm at the same place and time
- RNA is NOT processed
Eukaryotes
- transcription takes place in the nucleus
- transcript is processed (spliced) and then exported to the cytoplasm
- T and T take place in separate compartements
RNA polymerase
- separates DNA strands
- Catalyzes the addition of new rNTPs
- moves in 3’ to 5’ direction
- but!! newly synthesized RNA 5’ - 3’
- threads the newly created RNA through a channel
- does not bind directly to the promoter…
- made up of several protein subunits (quat. structure)
RNAP II
one of the tree forms of RNA polymerase in eukaryotes
transcribes genes for mRNA (and a few other RNAs such as small nuclear RNA, miRNA)
what happens in transcription?
- RNA POL binds to a region called a PROMOTER
- transcription starts at a position called the +1 site/transcription start site (transcribes the first base/nucleitide)
- transcription stops after the RNA POL passes through a region called the terminator
RNA polymerization steps
- incoming nucleotides are accepted if they correctly base pair with the template DNA
- the 3’-OH of the growing stand attacks the high energy phosphate bond of the incoming ribonucleotide, providing the energy to drive the reaction
- the two phosphates of the incoming ribonucleotide are released as pyrophosphate
holoenzyme
Sigma factor + RNA POL
Bacteria
moves in the direction from -10 box and +1 site, downstream
orientation of the sigma/RNA POL relative to the -35 and -10 box is important — the RNA complex “know” which direction to move along the strand
ability to bind tightly to the promoter is a level of control
general transcription factors bind where
bind to the promoter
where do transcriptional activator proteins bind
enhancers
Eukaryotes
imitation of transcription at a eukaryotic promoter
TATA binding protein (TBP) and general transcription factors (activators) band to the promoter to recruit the binding of RNA POL
Basal transcription complex
minimum complex needed to initiate transcription
TATA protein and General Transcription Factors (and activators) bound to the promoter
Eukaryotes
where does transcription start in eukaryotes vs bacteria
bacteria: +1 site (10 bases downstream from -10 box)
eukaryotes: transcription start ~25 bases downstream from the promoter
when does transcription stop
after RNA POL passes through the terminator
transcribed but not translated
end of transcription in eukaryotes vs bacteria
EUKARYOTE
- termination sequence is transcribed as well
- involves proteins called TERMINATION FACTORS (term not tested)
- binding of these termination factors to the DNA disturbs the RNA POL
BACTERIA
- transcribes through a termination sequence
- a hairpin loop forms on the mRNA, causing the RNA POL to dissociate from the GENE
DNA binding proteins
- bind in the major and minor grooves of DNA
- recognize specific sequences of base pairs
- bind by H-bonds and other non-covalent interactions interact with the bases
- how strongly a protein binds will determine how long it will stay on the DNA strand and whether transcription and/or translation will proceed
how can transcription be regulated
how OFTEN and how TIGHTLY RNA POL (and sigma or other transcription factors) bind to the promoter
binding more often and tightly results in more RNA transcripts made from that gene!!
5’ cap
modified base linked by its 5’ carbon to the 5’ end of the primary transcript by a bridge composed of 3 phosphates
basically a nucleotide added in reverse
Essential for translation because this is how the ribosome recognizes mRNA!!
3 chemical modifications of Eukaryotic Primary RNA transcript
- addition of 5’ cap
- polyadenylation (forming poly (A) tail)
- splicing of introns and exons
Exons
expressed regions
code for amino acids
sometimes removed
introns
intervening sequences
do not code for everything
always removed
splicing
cutting out introns
alternative splicing
- primary transcripts from the same gene can be spliced in different ways to yield different mRNA
- Allows the same transcript to be processed in diverse ways
- when cells also splice out some of the EXONS
- leads to 2 cells producing 2 proteins with similar function but varying number of AA found in the protein
5’ and 3’ UTRs untranslated regions
mRNAs contain regions before the start codon and after the stop codon
alternative splicing
different splicing pattern
creates splice variants
means that more than one type of protein can be made from a single gene coding for an mRNA
DNA packaging as transcriptional control
BACTERIA
- promoters are unpackaged
- accessible to RNA polymerase
- default state is always on
EUKARYOTES
- DNA is packaged tightly
- must be unpackaged for the RNA polymerase to access the DNA and start transcription
Primary transcript
RNA transcript that comes off the template DNA strand and contains the complement of every base that was transcribed from the DNA template
○ Includes the information needed to direct the ribosome to produce the protein corresponding to the gene
mRNA
RNA molecules that combines with the ribosome to direct protein synthesis, carries genetic message from DNA to ribosome
Primary transcripts for protein-coding genes in prokaryotes features not shared with those in eukaryotes:
primary transcripts are translated immediately
In prokaryotes, the primary transcript IS the mRNA
often contain genetic info for the synthesis of 2 or more different proteins
Polycistronic mRNA
molecules of mRNA that code for multiple proteins
RNA processing
chemical modification of the primary transcripts, converts it into the finished mRNA, which can then be translated by the ribosome
steps of RNA processing
- 5’ end of the primary transcript is modified by the addition of 5’ cap
- Polyadenylation – addition of a string of ~250 consecutive A-bearing ribonucleotides to the 3’ end, forming poly(A) tail
- Removal of non-coding introns
- Ribosomal RNA (rRNA)
○ Essential in translation
○ Genes and transcripts for rRNA are concentrated in the nucleolus
§ Non-membrane bound spherical structure observed within the nucleus
- Transfer RNA (tRNA)
○ Carries individual amino acids for use in translation
most abundant transcripts in mammalian cells
ribosomal RNA and transfer RNA
Needed in large amounts to synthesize the proteins enconded in mRNA
transcription bubble
where transcription takes place, strands of DNA duplex are separated
RNA-DNA duplex
the growing end of the RNA transcript is paired with the template strand
General process of polymerization of RNA
incoming ribonucleoside triphosphate is accepted by RNA polymeras only if it undergoes proper base pairing with the base in the template DNA strangd
- RNA polymerase orients the 3’ end of the growing strand so that the oxygen in the hydroxyl group can attack the innermost phosphate of the triphosphate of the incoming ribonucleoside, competing for the covalent bond
When cleaved, provides the energy to drive the reaction that creates the phosphodiester bond attaching the incoming nucleotide to the 3’ end of the growing chain
