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Erstellt von Marissa Alvarez
vor mehr als 5 Jahre
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Lecture 42 learning goals
Understand the following key points
Notes on gene orientation
• In double-stranded DNA, the top strand is written in ___, while the bottom strand is written in __ direction.
A gene starts at a point where ___ starts and is called 5'-end.
Moving along a gene in the 5' - 3' is called ____ while moving in the 3' - 5' is called ____.
Transcription start site of a gene in double-stranded DNA is written on
the __, while the termination site is written on the ___.
• In double-stranded DNA that codes for a gene, the top stand is called the ___ strand (___ strand) since its sequence corresponds to the transcribed RNA product.
On the other hand, the bottom strand is
called ___ strand (____ strand) since it serves as a ___ for gene transcription.
The enzymes that catalyze RNA synthesis are called RNA _____
E. coli RNA polymerase is composed
of ___ core polypeptides with a
stoichiometry of α2ββ'ω.
All ___ eukaryotic polymerases have
five core polypeptides homologous to
the E. coli RNA polymerase.
• Also, all eukaryotic RNA pols share
___ common subunits, and each
contains three to seven unique smaller
subunits.
Gene transcription by RNA pols
Unlike DNA polymerases, RNA polymerases can initiate RNA synthesis by ___ in the presence of initiation factors such as subunits in prokaryotes and the general transcription factors in eukaryotes.
• ____: DNA sequences in genes that often lie upstream of transcription start site and recruit RNA pols for transcription.
Promoter consensus sequences for different genes ___.
• Genes with __-rich consensus sequences are frequently transcribed (e.g. rRNA operon) and are considered strong promoters, while those with __-rich are less frequently transcribed (aka weak promoters).
Transcription initiation
RNA pol binds to the ___, unwinds DNA, induces transcription bubble formation, positions template at the active site, and starts transcription.
NTPs that diffuse to the active site base pair with the template at the +1
and +2 position, leading to the formation of a 3'-5' ___ bond.
RNA synthesis is almost
identical to DNA synthesis
Reaction catalyzed by RNA Pol.
___, not dNTPs are used
• Incoming NTP base pairs
• Nucleophilic attack by the _-__ group on
the α-phosphate of the NTP
• 3'-5' ____ bond formation
• Release of the βγ phosphate from the NTP as pyrophosphate (PPi).
• Hydrolysis of the PPi by ____, providing an additional thermodynamic driving force that favors the reaction.
• In E. coli, the rate of transcription is 30-85 NTPs per second, which is ___ than DNA synthesis.
Elongation of RNA synthesis
Following the formation of the first phosphodiester bond, RNA polymerase
"walks" along one strand of DNA, known as the ___ strand, in the
3' to 5' direction.
For each nucleotide in the template, RNA polymerase adds a matching (complementary) RNA nucleotide to the _ end of the RNA strand.
After adding about 10 nucleotides, RNA pol holoenzymes move away from the promoter region. This step is known as promoter ___.
Synthesis continues until the polymerase encounters a ____ signal.
Termination of RNA synthesis
There are two major types in bacteria: __-___ and ___-___
In _-___ termination, the RNA contains a binding site for a protein called Rho factor.
Rho factor binds to this sequence, travels
towards RNA polymerase, and induces ____, leading to the release of RNA.
The Rho factor is helped by the presence of a __-rich transcription-stop point in the DNA that pauses the RNA pol.
Rho-independent termination:
Close to the termination site, RNA has
complementary GC-rich two-fold symmetry sequence that folds back on itself and forms a stable ___ that causes the polymerase to ___.
The hairpin is followed by a series of _ nucleotides in the RNA, which makes weak base pairing with As in the DNA template, allowing ___ of the RNA transcript.
Coupled with the ___ polymerase, weak pairing causes ___ and
release of the new RNA transcript
Termination of transcription in EUKARYOTES
• More complex and not fully understood
• In case of mRNA transcription, the process involves appearance of __-_ signal sequence in the RNA transcript
• Steps in mRNA termination: binding of CPSF (cleavage and poly-A signal factor) to ___ signal sequence, cleavage of the nascent transcript, and addition of __-__ ___
RNA particularly tRNA, rRNA, and mRNA
are ____ before use in cells
Post-transcriptional tRNA processing
(a) RNase P and other ____
cleave the primary transcript
Post-transcriptional tRNA processing
(b) RNase __ trims the 3' end.
Post-transcriptional tRNA processing
(c) tRNA nucleotidyl transferase adds
___ to the 3' end.
rRNA processing
In prokaryotes, rRNA is produced as a single transcript and then cleaved
by the actins of __ __ ____ to mature 16S, 23S, and 5S.
Endonucleases M16, M23, and M5 trim the __ ___ to final mature forms.
• In eukaryotes also, three of the rRNAs are produced by cleavage of a ___ transcript — 18S, 5.8S and 28S are produced.
The fourth rRNA in eukaryotes, 5S, is produced as a monomer by __ ___ __ and is processed separately
Note: ___ unit (symbol S, sometimes Sv) is a non-metric unit for the sedimentation coefficient. The ___ unit (S) offers a measure of a particle's size based on its sedimentation rate, i.e. how fast a particle of given size and shape '___' to the bottom of a solution.
Eukaryotic rRNA processing
The 45S pre-rRNA is transcribed in the ___ and processed into the
mature 18S, 5.8S and 28S rRNAs by a series of ____ and ____ steps.
Note that methylation of regions that become mature rRNA occurs BEFORE removing the ___.
Messenger RNA processing
• In prokaryotes, translation begins even before transcription is ___.
• In eukaryotes transcription occurs in the ___ and translation occurs in the ___.
____ allows eukaryotes to process mRNA.
• In eukaryotes, primary mRNA transcripts are processed in the following ways:
1. 5'-end ____
2. 3'-end ____
3. Splicing ___ and removing ___
Capping of the 5'-end of mRNA
Formation of a cap at the 5' end of mRNA.
The 5' end of eukaryotic mRNA
is a nucleoside triphosphate initiated by RNA pol __. This end is capped to increase ___ and suitability for translation.
Steps
• Removal of the γ-phosphate by ____.
Capping with GTP by ___ ___ to create a 5'-5' GTP linkage
• ____ of the G at N-7 and a few successive 2' oxygen atoms of ribose.
Polyadenylation
Steps
The appearance of __-__ signal
Recognition of poly-A signal by ___ (Cleavage and Poly-A Signal Factor)
Polyadenylation by poly-A ___ and release
Note: Polyadenylation enhances mRNA ___ and transport to the cytoplasm
where translation takes place.
Splicing __ to produce mature mRNA
General scheme of splicing:
In eukaryotes, primary mRNA
transcripts are much larger, and mature ones are produced by what is known as
splicing of ___ and removal of ___.
mRNA Splicing
The splicing of two successive exons in a nascent mRNA transcript requires removal a single ___, using two ____ reactions:
1. between the 5' splice site and the branch site adenylate
AND
2. between the 5' exon and the 3' splice site
The products of these reactions are ___ exons and an ___ intron
These reactions are catalyzed by a large RNA-protein complex called the
___ composed of over 100 proteins and 5 RNA molecules.
The RNA molecules are called ___ __ ___ (__) and when they associate with proteins they are called small nuclear ribonucleoproteins (snRNAPs) and is sometimes referred to as ___.
The five snRNAs are named U1, U2, U4, U5, and U6 (U stands for ___ which is common in the snRNAs).
(a) The spliceosome positions the adenylate residue at the branch site near the __ splice site. The 2'-hydroxyl group of the adenylate attacks the __ splice site.
(b) The 2'-hydroxyl group is attached to the 5' end of the ___, and the newly created 3'-hydroxyl group of the exon attacks the __ splice site.
(c) As a result, the ends of the exons are joined, and the intron, a ___-shaped molecule, is ___.
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