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Lecture 42 - RNA transcription and processing
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- 1Lecture 42 learning goals Understand the following key points• RNA polymerases — they are complex multi-subunit enzymes
• Genes — have coding (sense) and template (anti-sense) strands
• Gene promoters — sites for initiation of transcription
• Mechanism of transcription initiation — separation of strands at promoter sites
• Mechanism of RNA synthesis — nucleotide addition
• Mechanism of transcription elongation and termination
• RNA processing - RNA, rRNA, mRNA - 2Notes 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.5' - 3'
3' - 5'
transcription
downstream
upstream
left
right
coding (sense)
template (antisense)
template - 3The 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.polymerases
five
three
four
- 4Gene 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).themselves
Promoter
vary
AT
GC - 5Transcription 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.promoter
phosphodiester
- 6RNA 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.NTPs
3'-OH
phosphodiester
pyrophosphatase
slower - 7Elongation 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.template
3'
clearance
termination
- 8Termination 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-dependent and Rho-
independent.
Rho-dependent
disengagement
GC
- 9Rho-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 transcripthairpin
stall
U
separation
stalled
disengagement
- 10Termination 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 __-__ ___poly-A
poly-A
poly-A tail - 11RNA particularly tRNA, rRNA, and mRNA are ____ before use in cellsprocessed
- 12Post-transcriptional tRNA processing (a) RNase P and other ____ cleave the primary transcriptendonucleases
- 13Post-transcriptional tRNA processing (b) RNase __ trims the 3' end.D
- 14Post-transcriptional tRNA processing (c) tRNA nucleotidyl transferase adds ___ to the 3' end.CCA
- 15rRNA 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.Rnase Ill endonuclease
three rRNA
single
RNA pol Ill
Svedberg
Svedberg
settles
- 16Eukaryotic 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 ___.nucleolus
endonucleolytic and exonucleolytic
introns
- 17Messenger 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 ___complete
nucleus
cytoplasm
Compartmentalization
capping
polyadenylation
splicing exons and removing introns - 18Capping 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.ll
stability
phosphohydrolase
guanylyl transferase
Methylation - 19Polyadenylation 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.poly-A
CPSF
polymerase
stability
- 20Splicing __ 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 ___.exons
exons
introns
- 21mRNA 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).intron
transesterification
joined
excised
spliceosome
small nuclear RNA (snRNA)
snurps
uracil
- 22(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 ___.5'
5'
intron
3'
lariat
released