RNA: Synthesis, Processing & Modification Flashcards
DNA undergoes
Replication, Repair and Genetic Recombination “RRG”
Flow of Genetic Information is based on the
Central Dogma of Molecular Biology
RNA synthesis undergoes
Transcription
Protein synthesis undergoes
Translation
Is a polymer composed of alternating units of ribonucleotides connected through a ____________.
RNA. 3’-5’ phosphodiester bond.
In contrast with DNA, ribonucleotides contain: _________ on the 2’-carbon of the ribose sugar. The base is ______ in place of thymine.
Hydroxyl. Uracil.
Significance of RNA: the __________ of DNA. Expresses the ______ contained in DNA.
Working copies. Master plan.
Major RNA classes: 80% of the total RNA.
Ribosomal RNA (rRNA)
Major RNA classes: smallest of the 3 major RNA species (excluding the _________), make up 15%.
Transfer RNA (tRNA)
Major RNA classes: 2-5%, but it is the most heterogenous in terms of size and base sequence.
Messenger RNA (mRNA)
Major RNA classes: are involved in mRNA splicing and gene regulation.
Small RNAs
Class of Eukaryotic RNA: Stable. less than 1% of total abundance. 100’s-1000’s.
Micro (miRNA)
Class of Eukaryotic RNA: Very stable. 1% of total abundance. 30 different species.
Small nuclear (snRNA)
Class of Eukaryotic RNA: Very stable. 15% of total abundance. 60 different species.
Transfer (tRNA)
Class of Eukaryotic RNA: Unstable to very stable. 2-5% of total abundance. 10 to the 5th different species.
Messenger RNA (mRNA)
Class of Eukaryotic RNA: Very stable. 80% of total abundance. 28s, 18s, 5.8s & 5s.
Ribosomal (rRNA)
Svedberg units for Prokaryotic rRNA
23s, 16s & 5s
Svedberg units for Eukaryotic rRNA
28s, 18s, 5,8s & 5s
“S” refers to _________, which is a measure of size based upon the molecular sedimentation rate during ultracentrifugation.
Svedberg Unit
Similarities between DNA & RNA synthesis: both have the general steps of _______, ______ & _______ with 5’ to 3’ polarity (synthesized in a 5’ to 3’ direction, antiparallel to the DNA template strand which is read in a 3’ -> 5’ direction)
Initiation, Elongation & Termination
Similarities between DNA & RNA synthesis: Both have large, _________ initiation complexes. Both adhere to ________ base-pairing rules.
Multicomponent. Watson-Crick.
Differences between DNA & RNA synthesis: _____ used in RNA synthesis rather than deoxyribose.
Ribose
Differences between DNA & RNA synthesis: ______ replaces thymine as the complementary base pair amino acid in RNA.
Uracil
Differences between DNA & RNA synthesis: A ______ is not involved in RNA synthesis.
Primer
Differences between DNA & RNA synthesis: only a portion of the genome is transcribed or copied into ____, whereas the entire genome must be copied during ____ replication.
RNA. DNA.
Differences between DNA & RNA synthesis: there is no ___________ function during RNA transcription.
Proofreading.
Is always read in the 3’ to 5’ direction in RNA.
Template strand
The opposite strand is called the
Coding strand
The enzyme responsible for the polymerization of ribonucleotides into a sequence complementary to the template strand of the gene. The enzyme attaches to the promoter on the template strand. This is followed by the initiation of ________ at the starting point, and the process continues until a termination sequence is reached.
DNA Dependent RNA Polymerase. RNA synthesis.
General features of genes: the starting point of transcription corresponds to the ________ of the mRNA. This is designated position ____, as is the corresponding nucleotide in the DNA. The numbers increase as the sequence proceeds ______.
5’ nucleotide. +1. Downstream.
General features of genes: The nucleotide in the _______ adjacent to the transcription initiation site is designated ____, and these negative numbers increase as the sequence proceeds _______, away from the initiation site.
Promoter. -1. Upstream.
Is defined as the region of DNA that includes the signals for transcription. Involves ________, _______ and _______.
Transcription unit. Initiation, Elongation & Termination.
Includes a promoter, an RNA coding region and a terminator.
Transcription unit
The RNA product, which is synthesized in the 5’ to 3’ direction is the
Primary transcript
A core enzyme, with its 4 subunits (plus an _____ subunit which is not shown), responsible for 5’->3’ RP activity. This enzyme lacks specificity.
Prokaryotic RNA Polymerase
Cannot recognize the promoter on the DNA template
Lacks specificity
Contains the sigma subunit or sigma factor that enables RNA polymerase to recognize promoter regions on the DNA. (Sigma + core enzyme). This is involved in the transcription by Prokaryotic RNA Polymerase.
Holoenzyme
An auxiliary protein of RNA polymerase. Some regions of DNA that signal the termination of transcription are recognized by RNA polymerase itself. Others are recognized by specific termination factors, an example of which is the ________ of E.coli.
Termination factor. rho (p) factor.
The primary transcripts generated by _________ (one of three nuclear DNA- dependent RNA polymerase in eukaryotes) are promptly capped by _________.
RNA Polymerase II. 7-methylguanosine triphosphate.
Are necessary for the subsequent processing of the primary transcript to mRNA, and for protection of the mRNA exonucleolytic attack.
Caps made by 7-methylguanosine triphosphate
Involves binding of the RNA polymerase holoenzyme to a promoter region.
Initiation
Pribnow box and -35 sequence. Highly conserved. Recognized by prokaryotic RNA polymerase sigma factors.
Consensus Nucleotide Sequences
A stretch of 6 nucleotides (__________) centered around 8 to 10 nucleotides to the left of the transcription start site that codes for the initial base of mRNA.
Pribnow box. 5’-TATAAT-3’.
A second consensus nucleotide sequence centered around 35 bases to the left of the transcription start site.
-35 sequence
A mutation in either the Pribnow box or -35 sequence can affect the _______ of the gene controlled by the ________.
Transcription. Mutant promoter.
Once the promoter has been recognized by the holoenzyme, __________ begins to synthesize a transcript of the DNA sequence (usually beginning with a ______), and the sigma subunit is released.
Elongation. RNA Polymerase (RNAP). Purine.
Unlike DNA polymerase, RNA polymerase does not require a _______ and has no _____ or _______ activity. (No ______ capability)
Primer. Endo or exonuclease. Repair.
General mechanism of RNA synthesis: 1. ________ by addition of ribonucleotides to the 3’-OH end.
Elongation
General mechanism of RNA synthesis: 2. acts as a nucleophile, attacking the a-phosphate of the incoming ribonucleoside triphosphate and releasing ________.
3’-OH. Pyrophosphate.
General mechanism of RNA synthesis: 3. Mechanism is the same as that used for elongation of a ________.
DNA strand
Signal is reached
Termination
May be required for the release of the RNA product.
Rho factor
Alternatively can recognize termination regions on the DNA template
Tetrametric RNA Polymerase
RHO Dependent Termination: requires _______ protein. It binds to a _______ near the 3’ end of the newly synthesized RNA, and migrates along behind the _______ in the 5’ to 3’ direction until the termination site is reached.
Rho factor. C-rich region. RNA polymerase.
RHO Dependent Termination: Rho factor has ________ RNA-DNA _______ activity that hydrolyzes ATP, and uses the energy to unwind the 3’ end of the transcript from the template.
ATP-dependent. Helicase.
RHO Dependent Termination: At the termination site, rho factor displaces the ___________, facilitating the dissociation of the RNA molecule.
DNA template strand
RHO Dependent Transcriptional Termination: 1. ________ transcribes DNA.
RNA polymerase
RHO Dependent Transcriptional Termination: 2. ____ attaches to its recognition site on _____.
Rho. RNA.
RHO Dependent Transcriptional Termination: 3. Rho moves along RNA, following the polymerase. _________ pauses at terminator and ____ catches up.
RNA polymerase. Rho.
RHO Dependent Transcriptional Termination: 4. Rho ______ the DNA RNA hybrid in the ________.
Unwinds. Transcription bubble.
RHO Dependent Transcriptional Termination: 5. ________. RNA polymerase, rho and RNA are released.
Termination
RHO- independent Termination: requires that the newly synthesized RNA have a stable ________ that slows down the progress RNA polymerase and causes it to pause temporarily.
Hairpin turn
RHO- independent Termination: requires that the newly synthesized RNA have a hairpin turn ________ to a region of the _______ near the termination region that exhibits 2-fold symmetry as a result of the presence of a _______.
Complementary. DNA template. Palindrome.
Transcription of Eukaryotic Genes: in addition to _____ recognizing the promoter region and initiating RNA synthesis, several supplemental ____________ bind to DNA in eukaryotes.
RNAP. Transcription Factors (TFs).
Transcription of Eukaryotic Genes: For RNAP and the TFs to recognize and bind to the specific DNA sequence, the ________ must assume a loose conformation and dissociate temporarily from nucleosome core.
Double helix.
Most actively transcribed genes are found in a relaxed form of chromatin.
Euchromatin
Most inactive segments are in a highly condensed
Heterochromatin
The interconversion of active and inactive forms of chromatin is called
Chromatin modeling
Genes that are inactive contain more
Methylated DNA (5-methylcytosine)
When histones become ________, the chromatin structure becomes looser.
Acetylated
High sensitivity to alpha-Amanitin. Major products are mRNA (nucleoplasm), miRNA, viral RNA.
RNA Polymerase II
Insensitive to alpha-Amanitin. Major products are 28s, 18s, 5.8 s in the nucleotides.
RNA Polymerase I
Intermediate sensitivity to alpha-Amanitin. Major products are tRNA/55 rRNA & snRNA
RNA Polymerase III
Eukaryotic RNA Polymerases: require additional proteins called ________ in order to specifically bind to a promoter and initiate transcription.
Transcription factors
Eukaryotic RNA Polymerases: are composed of a variety of different ___ sequence elements which recruit some of these ________ factors through DNA-protein interactions.
Eukaryotic promoters. Cis. Trans-acting.
Eukaryotic RNA Polymerases: also occur and account for many of the multi component complexes found at eukaryotic promoters.
Protein-protein interactions
Eukaryotic RNA Polymerases: utilize a small number of ubiquitous transcription factors while ____ uses a large variety of specific ones.
Pol I & III. Pol II.
Initiation at RNA Pol I promoters: 1. 2 identical subunits of the Upstream Binding Factor bind to the ___________ and the _________.
Upstream Core Element. Core Promoter Element.
Initiation at RNA Pol I promoters: 2. protein-protein interactions between ________ force these two DNA sequences to come into close proximity.
UBF molecules
Initiation at RNA Pol I promoters: 3. This enables subsequent binding of _________, which consists of four subunits.
Selectivity Factor I
Initiation at RNA Pol I promoters: 4. Ultimately, this stabilized structure permits binding of other factors and finally _______.
RNA Polymerase I
Initiation at RNA Pol III promoters: Initiation of transcription of a tRNA gene: 1. The _______ transcription factor binds via a recognition of the A and B sites.
TFIIIC
Initiation at RNA Pol III promoters: 2. This permits subsequent binding of the trimeric ______ immediately upstream of the transcription start site.
TFIIIB
Initiation at RNA Pol III promoters: 3. In response to TFIIIB binding, _________ is recruited and initiates transcription.
RNA Polymerase III
Initiation at RNA Pol III promoters: In the case of 5S tRNA genes, the process is similar, except that an additional factor, _____, is required. TFIIIA binds the ______, which permits subsequent binding of TFIIIB and TFIIIC, then recruitement of RNA Polymerase III.
TFIIIA. C box.
Promoters for the Class II Genes: A sequence DNA nucleotides that is almost identical to the Pribnow box is usually found centered about __________ upstream of the initial base of the transcription start site for an mRNA molecule. This consensus sequence is the _____________. The consensus sequence of this element is ________(so it resembles the TATAAT sequence of the prokaryotic -10 region)
25 nucleotides. TATA or HOGNESS BOX. TATAAAA.
Promoters for the Class II Genes: appears to be more important for selecting the start point of transcription (i.e. Positioning the enzyme) than for defining the promoter.
TATA box
Promoters for the Class II Genes: _____, which binds to the TATA box promoter element, is the only transcription factor capable of binding to specific sequence of DNA. It consists of _________ and _________.
TFIID. TATA binding protein (TBP) and 14 TBP-associated factors (TAFs).
Promoters for the Class II Genes: A second consensus called the ________(70-80 nucleotides) is found upstream from the transcription start site.
CAAT box
Promoters for the Class II Genes: The transcription factor ___ or ___ binds to CAAT box.
CTF or NF1.
Promoters for the Class II Genes: The ______ is a common element in eukaryotic class II promoters. Its consensus sequence is ________.
GC box. GGGCGG.
Promoters for the Class II Genes: the _________ binds to the GC box.
Transcription factor Sp1
Enhancers: Special _________ sequences that increase the rate of initiation of initiation of transcription by ______. Must be on the same chromosome as the gene whose transcription they stimulate. They can be located ______ (to the 5’ side) or ______ (to the 3’ side) of the transcription start site.
Cis-acting DNA. RNAP II. Upstream. Downstream.
They can be close to or thousands of base pairs away from the promoter. They can occur on either strand of the DNA.
Enhancers
Enhancers contain DNA sequences called ________ that bind specific transcription factors called ________.
Response Element. Activators.
Enhancers: By _______ or _______ the DNA, enhancer-binding factors can interact with transcription factors bound to a promoter and with RNAP II, stimulate transcription.
Bending or Looping.
Enhancers: are similar to enhancers in that they act over long distances to reduce the level of gene expression.
Silencers
Formation of PIC for Pol II on a TATA promoter: recognizes and binds the TATA box.
TFIID
Formation of PIC for Pol II on a TATA promoter: binds and stabilizes TFIID binding.
TFIIA
Formation of PIC for Pol II on a TATA promoter: assembles ppssibly in a stepwise manner to form a preinitiation complex.
RNA Polymerase II Holoenzyme
Formation of PIC for Pol II on a TATA promoter: Finally, the various _______ bind to complete formation of the pre-initiation complex.
Regulatory (Srb-Mediator, Srb10-CDK and Swi-Snf)
Differs from its prokaryotic counterpart in that it has a series of heptad repeats with consensus sequence Try-Ser-Pro-Thr-Ser-Pro at the carboxyl terminal of the largest pol II subunit.
Eukaryotic Pol II
Is both a substrate for several kinases, including the kinase component TFIIH, and a binding site for a wide array of proteins.
Carboxyl Terminal Repeat Domain (CTD)
Are the ultimate targets of cell-signaling pathways. Whenever cells need to respond to an extracellular signal (ex.hormone) the response is mediated by a change in gene expression that comes about, most often as the result of a change in the phosphorylation state of a transcription of factor.
Transcription factors
Inhibits transcription initiation by binding to the beta subunit of prokaryotic RNA polymerase, preventing phosphodiester bond formation.
Rifampin
Is used for tumor therapy. It binds to the DNA template and interferes with the movement of RNA polymerase along the DNA.
Doctinomycin (Actinomycin D)
Transcription Inhibitors: intercalate between successive G=C base pairs in duplex DNA. Inhibit transcriptional elongation in pro and eukaryotes.
Actinomycin D. Acridine.
Transcription Inhibitors: binds with B-subunit of bacterial RNA polymerase. Blocks promoter clearance. (Elongation)
Rifampicin
Produced by fungus Amanita phalloides (death cap mushroom). Potent inhibitor of RNA pol II and weak inhibitor of RNA pol III.
a-Amanitin
Is the linear copy of the transcription unit.
Primary transcript
The primary transcripts of both prokaryotic and eukaryotic RNA are post-transcriptionally modified by cleavage using
Ribonucleases
Are further modified to help give each species a unique identity.
tRNAs
Is generally identical to its primary transcript
Prokaryotic mRNA
Is extensively modified after transcription.
Eukaryotic mRNA
For prokaryotes and eukaryotes. Synthesized from preribosomal RNA.
Ribosomal RNA
Are produced from a single RNA precursor molecule which is processed in the nucleolus. Same as eukaryotic rRNA 28s, 18s & 5.8s
23s, 16s and 5s ribosomal RNA of prokaryotes
Is synthesized by RNAP III and modified separately.
Eukaryotic 5S rRNA
Are cleaved by ribonucleases to yield intermediate-sized pieces of rRNA, which are further trimmed to produce the required rRNA species.
Preribosomal RNAs
Transfer RNA: 1. tRNA is transcribed by ________. The transcription product, the ______, contains additional RNA sequences at both the 5’ and 3’ ends. These additional sequences are removed from tye transcript during processing. The additional nucleotides at the 5’ end are removed by an unusual RNA containing enzyme called _________.
RNA Polymerase III. pre-tRNA. Ribonuclease P (RNase P)
Transfer RNA: 2. Some tRNA precursors contain an _______ located in the anticodon arm. These are spliced out during processing of the tRNA.
Intron
Transfer RNA: 3. ________ these three bases are not coded for by the tRNA gene. Instead, these nucleotides are added during processing of the pre-tRNA transcript (by replacing UU residues at the 3’ end) The enzyme responsible for the addition of the CCA-end is _________.
CCA at the 3’-end. tRNA nucleotidyl transferase.
Transfer RNA: 4. _________ are introduced into the tRNA at the final step. Addition of ________, ________ and _______ bases. Modification of anticodon arm.
Base modifications. Dihydrouridine, Pseudouridine & Methylated.
Eukaryotic mRNA: The RNA molecule formed by RNAP II (the primary transcript) contains the sequences found in _________.
Cytosolic mRNA
Eukaryotic mRNA: The primary transcripts are modified in the
Nucleus
Eukaryotic mRNA: The collection of all the precursor molecules for mRNA is known as
Heterogenous Nuclear RNA (hnRNA)
5” Capping: _________ is attached to the 5’ end of the mRNA. GTP addition is catalyzed by __________ in the nucleus. This followed by the _________ of terminal guanine in the cytosol, catalyzed by ___________.
7-Methylguanosine. Guanylyl transferase. Methylation. Guanine-7-Methyltransferase.m
Is the source of methyl group.
SAM
Permits the initiation of translation and helps stabilize mRNA.
Cap addition
Eukaryotic mRNA with _________ are not efficiently translated.
No cap
Most eukaryotic mRNAs have a chain of 40 to 200 adenine nucleotides to the 3’ end.
Poly-A-Tail
Poly-A-Tail: not transcribed from DNA but added by the nuclear enzyme _________.
Polyadenylate Polymerase
Poly-A-Tail: A consensus sequence (_________) found near the 2’ end signals that a _________ must be added to stabilize RNA and facilitate its exit from the nucleus. After entering the ________, the poly a tail is shortened.
AAUAAA. Poly-A-Tail. Cytosol.
Removal of RNA sequences that do nit code for proteins (intervening sequences) from the primary transcript.
Removal of Introns
The remaining _____ are spliced together by ______ to form mature mRNA.
Exons. Spliceosome.
The intron loops out as _______(______________ which are complexes of snRNAs and proteins) bind to form the spliceosome.
snRNPs. Small nuclear ribonucleoprotein particles or snurps.
The intron is _______ and the exons are then _______ together. The excised intron is released as a “_________” structure and degraded.
Excised. Spliced. Lariat.
The resulting _________ may then exit the nucleus and be translated in the ________.
Mature mRNA. Cytoplasm.
Removal of introns from pre-mRNA transcripts involves cleavage at the 5’-end of the intron by attack of a specific _________, the ________.
2’OH group. Branch site.
This forms of a phosphodiester bond with the 5’-phosphate of the Intron, creating a ________.
Lariat structure
The intron lariat is then removed, proceeding by attack of _____ on exon 1 to displace the intron from the 5’-phosphate of exon 2. The ____ and _____ sequences at the branch site are variant.
3’-OH. GU & AG.
The fatal disease which results from an autoimmune response where the patient produces antibodies against host proteins, including ______.
SLE. snRNPs.
Mutations that cause the incorrect splicing of beta-globin mRNA are responsible for some cases of
Beta-thallasemia
