BCEM 393 - Transcription, RNA Processing, and Translation

Fill in the blanks: the synthesis of RNA from a DNA template is transcription and it requires the following to happen: __________, _________, _________, and ____.

Template DNA, NTPs (ATP, CTP, GTP, UTP), RNA polymerase, Mg²⁺

Fill in the blanks: the three general steps of transcription are __________, ___________, and __________.

Initiation, elongation, termination

Fill in the blanks: RNA polymerase has the subunits ____ (for assembly of core enzyme, and interacts with regulatory factors), ____ (for catalysis and interactions with DNA and RNA), ____ (required for structure/folding), and _____ (for promoter recognition).

α₁α₂, β and β’, ω, α⁷⁰

Fill in the blanks: in E. coli, RNA polymerase is a very large (~500 kDa) and complex enzyme consisting of five kinds of subunits. The subunit composition of the entire enzyme, called the ___________, is α₂ββ’σω. RNA polymerase without this subunit is called the ___________, which contains the __________.

Holoenzyme, core enzyme, active site

Fill in the blanks: in RNA polymerase, the σ subunit initiates transcription in E. coli by recognizing and binding the ___________, and ___________ the affinity of RNA polymerase for DNA.

Promoter sequence, decreases

True or False: shortly after initiation of transcription, the σ subunit is lost, enabling the core enzyme to bind strongly to the DNA template.

True

Fill in the blank: elongation in E. coli begins after the formation of the first ____________.

Phosphodiester linkage

Fill in the blank: in contrast with DNA synthesis, RNA synthesis can start __________.

De novo, without needing a primer

Fill in the blanks: in the elongation reaction for RNA transcription, the newly synthesized RNA forms a hybrid helix with about 8bp of the template DNA strand. The ________ group of the RNA in this hybrid helix is positioned so that it can attack the _______ group of the incoming ___________.

3’-OH, inner most phosphoryl, ribonucleoside triphosphate

Fill in the blanks: a __________ followed by several ___________ in the RNA product causes RNA polymerase to pause transcription. The __________ base pairs are weak, so RNA dissociates from the DNA template and enzyme.

Hairpin, uracil residues, rU-dA

Fill in the blanks: RNA polymerase needs no help terminating transcription when it encounters a hairpin followed by several U residues. At other sites, however, termination requires the participation of an additional protein with _________ activity called ________. This type of termination is called _______________.

ATPase, rho (ρ), protein-dependent termination

True or False: the transcription reaction NTP + RNA_n + H₂O → RNA_n+1 + 2P_i is the same in prokaryotes and eukaryotes.

True

True or False: the 3’-OH group of the growing RNA chain attacks the innermost phosphate of the next NTP during initiation of transcription in both prokaryotes and eukaryotes.

True

True or False: RNA is formed in the 5’ → 3’ direction during transcription in both prokaryotes and eukaryotes.

True

True or False: transcription and translation are separated in time and space in both prokaryotes and eukaryotes.

False; transcription and translation are separated in time and space in eukaryotes, but can occur at the same time in prokaryotes

True or False: the transcription termination signal is a GC rich hairpin-poly(U) structure in both prokaryotes and eukaryotes.

False; the termination signal in prokaryotes is the GC rich hairpin-poly(U) structure. The termination signal in eukaryotes is the poly(A) tail

True or False: mRNA is highly processed and transported across the nuclear envelope after transcription in both prokaryotes and eukaryotes.

False; mRNA is highly processed and transported across the nuclear envelope in eukaryotes, and is not transported across a membrane in prokaryotes

Fill in the blanks: the promoter elements for eukaryotic RNA polymerase II include a __________ (located at approximately -25), _________ (initiator element, +1), __________ (downstream core promoter element, +30), __________ (can be > 1 kb from start site), and _____________ (-40 to -150).

TATA box, initiator element (Inr), downstream core promoter element (DPE), enhancer, CAAT box and GC box

Fill in the blank: promoters are referred to as __________ because a promoter and the gene it regulates are always on the same molecule of DNA.

Cis-acting elements

Fill in the blanks: in eukaryotes, transcription factors bind __________ to recruit RNA polymerase II. The TATA box is recognized by ________ by the _________. This initiates the formation of the ___________.

Cis-acting elements, transcription factor II D (TFIID), TATA binding protein (TBP), preinitiation complex (PIC)

Fill in the blanks: in eukaryotes, the pre-initiation complex (PIC) is formed in the following steps:

1. ___________ binds DNA and the ___________ moves between multiple conformations until it binds the __________.

2. ___________ binds and stabilizes the interactions between the complex and promoter region.

3. ___________ fully engages the promoter, bending the DNA.

4. ___________ recognizes ___________ and recruits RNA polymerase II and __________, and __________.

5. __________ unwinds DNA and phosphorylates the C-terminal domain of Pol II, triggering transition to elongation and recruitment of RNA processing enzymes.

Transcription factor II D (TFIID), TATA binding protein (TBP) domain, TATA box, TFIIA, TBP, TFIIB, TBP/DNA complex, TFIIF, TFIIE, TFIIH (a helicase)

True or False: in eukaryotes, the pre-initiation complex (PIC) initiates transcription at high levels. Additional transcription factors that bind to other sites stimulate low levels of transcription.

False; the pre-initiation complex (PIC) initiates transcription at low levels. Additional transcription factors that bind to other sites stimulate high levels of transcription

True or False: one transcription factor has little influence, many are needed to form a complex that stimulates or suppresses transcription.

True

Fill in the blank: one transcription factor has little influence, many are needed to form a complex that stimulates or suppresses transcription. This is called ___________.

Combinatorial control

True or False: prokaryotic and eukaryotic transcription have the same elongation reaction, but differ in the termination elements, regulation, and space/location.

True

True or False: prokaryotic transcription is regulated by transcription factors.

False; eukaryotic transcription is regulated by transcription factors

Multiple Choice: which of the following is true?

a) In eukaryotes, transcription happen simultaneously with translation

b) In prokaryotes a polyA tail terminates transcription

c) In eukaryotes, the elongation reaction uses a different mechanism from in prokaryotes

d) In prokaryotes the control elements are less complex

d)

Multiple Choice: what enzymatic activity do subunits of TFIIH have (select all that apply)?

a) Phosphatase

b) Kinase

c) Helicase

d) Ligase

b), c)

True or False: virtually all of the initial products of transcription are further processed in eukaryotes.

True

Fill in the blanks: in eukaryotes, primary transcripts (pre-mRNA molecules) acquire a __________ at their 5’ end and a __________ at their 3’ end.

7-methylguanasine cap, polyadenylate tail

Fill in the blanks: most genes in eukaryotes are discontinuous mosaics of regions that encode amino acids, called _______, and noncoding regions, called ______, that are removed by splicing.

Exons, introns

Fill in the blanks: during mRNA processing, introns, _________ regions, are ____________, and exons, __________ regions, are ____________.

Non-coding, removed, coding, joined during splicing

True or False: mRNA processing with the 5’ cap begins very early during transcription after about 20-30 nucleotides of RNA are synthesize.

True

Fill in the blanks: the 5’ cap in mRNA processing is made by the _____________, which is composed of ___ enzymes associated with phosphorylated RNA polymerase II.

Cap synthesizing complex, 4

Fill in the blanks: the 5’ cap protects from ______________, and interacts with ____________ to ______________.

Degradation by nucleases, ribosomes, enhance translation

Multiple Choice: the 5’ end of an unprocessed RNA transcript contains a:

a) 5’ triphosphate group of the first nucleotide

b) 5’ diphosphate group of the first nucleotide

c) 5’ phosphate group of the first nucleotide

d) 5’ OH of the first nucleotide

a)

Fill in the blanks: the 5’ cap modification of pre-mRNA occurs through the following steps:

1. The terminal ___________ at the 5’ end is removed by a ____________.

2. The ___________ 5’ end attacks the ___________ of a ____ to form a ____________ linkage.

3. The __________ atom of the terminal ___________ is then methylated.

4. The _____ of the adjacent ribose is methylated.

Phosphate, phosphohydrolase, diphosphate, α-phosphate, GTP, 5’ to 5’ triphosphate, N7 nitrogen, guanine, 2’OH

Fill in the blanks: the poly(A) tail in mRNA is about ____ ___________ at the ___ end of the RNA.

250, adenylates, 3’

Fill in the blanks: enzymes involved in building the poly(A) tail on mRNA are associated with the ______________ of __________.

Phosphorylated C-terminal domain, RNA polymerase II

Fill in the blanks: the role of the poly(A) tail on mRNA is to ___________ and ___________. It is also involved in ___________.

Enhance stability, enhance translation efficiency, nuclear export

Fill in the blanks: the poly(A) tail modification of pre-mRNA occurs through the following steps:

1. ___________ recognizes the ___________ and an endonuclease cleaves the mRNA transcript.

2. ____________ adds about ____ __________ to the ___ end.

Cleavage and polyadenylation specificity factor (CPSF), cleavage/polyadenylation signal, poly(A) polymerase, 250, adenylates, 3’

True or False: exons, non-coding regions, are removed and introns, coding regions, are joined together.

False; introns, non-coding regions, are removed and exons, coding regions, are joined together

Fill in the blanks: to achieve the accurate removal of introns, the correct splice site must be clearly marked. In eukaryotes, these sequences have structural motif: the intron begins with _____ and ends with ______.

GU, AG

Fill in the blanks: the __________ is a complex of protein and RNA that excises introns and joins exons.

Spliceosome

Define: transesterification.

The reaction of an alcohol and ester to make a different alcohol and ester

Fill in the blanks: in mammalian cells, the first steps of mRNA splicing (before spliceosome assembly) occurs through the following steps:

1. The ____________ attacks the phosphoryl at the ___ splice site. _________ is released and a __________ intermediate is formed.

2. The ____ of _________ attacks the phosphoryl group at the ____ splice site, giving the spliced product _________ and a ___________.

Branch site adenylate 2’OH, 5’, exon1, lariat, 3’OH, exon1, 3’, exon1-exon2, lariat intron

Fill in the blanks: the spliceosome is made of about _____ proteins and five critical __________; ____, ____, ____, ____, and ____.

300, small nuclear RNAs (snRNAs), U1, U2, U3, U4, U5

Fill in the blank: small nuclear RNAs (snRNAs) and proteins form ___________.

Small nuclear ribonucleoproteins (snRNPs)

Fill in the blanks: assembly of the spliceosome in mRNA splicing occurs through the following steps:

1. ____ binds the ____ splice site, initiating spliceosome assembly on the pre-mRNA molecule.

2. ____ binds the __________ in the _________.

3. The ____________ complex joins, and displaces _____ and _____. Extensive interaction between _____ and _____ brings the _____ splice site and _________ close together.

4. A first _________ is facilitated by the _________ of the spliceosome.

5. A second _________ is facilitated by _____. _____ is required for catalysis.

6. The products _________ and ________ are formed.

U1, 5’, U2, branch site, intron, U4-U5-U6, U1, U4, U2, U6, 5’, branch site, transesterification, catalytic core, transesterification, U5, Mg²⁺, spliced RNA products, lariat intron

Multiple Choice: which subunit of E. coli RNA polymerase contributes to decreased affinity of the enzyme for DNA?

a) β

b) ω 

c) α

d) σ

d)

Fill in the blanks: the genetic code links _________ and _________ information.

Nucleic acid, protein

Fill in the blanks: the genetic code is __________. This means that some amino acids are encoded by more than one codon, inasmuch as there are 64 possible triplets and only 20 amino acids. Codons that specify the same amino acid are called __________.

Degenerate, synonymous

Fill in the blanks: the start codon is ___, for the amino acid __________, and the stop codons are ___, ___, and ___.

AUG, methionine, UAA, UAG, UGA

Fill in the blank: _________ serves as the adapter molecule between the codon and its specified amino acid.

Transfer RNA (tRNA)

Fill in the blanks: all known tRNA molecules have the following features:

• Each is a single strand containing between __ to __ ribonucleotides (~25 kDa).

• The 3D molecule is __-shaped.

• The 5’ end is ____________.

• The activated amino acid is attached to a hydroxyl group of the adenosine residue located at the end of the __________ of the acceptor stem. This region is a flexible single strand at the 3’ end of mature tRNAs.

• The anticodon is present in a _________ near the center of the sequence.

73, 93, L, phosphorylated, 3’ CCA component, loop

Fill in the blank: tRNAs contain modified bases. For example, __________ is formed by deamination of adenine.

Inosine

Fill in the blanks: inosine, a modified base in tRNA, is formed by _________ of __________.

Deamination, adenine

Fill in the blank: in anticodons, I (e.g., IGC) is for __________.

Inosine

True or False: tRNAs only recognize one codon.

False; some tRNAs recognize more than one codon because of wobble base pairing

Fill in the blanks: when inosine (I) is at the 5’ end of the anticodon, the anticodon can recognize ___ codons because there is some _________ in the pairing of the ________ base of the codon. When U or G are at the 5’ end of the anticodon, the anticodon can bind ___ codons.

3, steric freedom (wobble), third, 2

Fill in the blank: when the first base of the anticodon is C, it can recognize ______ in the third base of the codon.

G

Fill in the blank: when the first base of the anticodon is A, it can recognize ______ in the third base of the codon.

U

Fill in the blank: when the first base of the anticodon is U, it can recognize ______ in the third base of the codon.

A or G

Fill in the blank: when the first base of the anticodon is G, it can recognize ______ in the third base of the codon.

U or C

Fill in the blank: when the first base of the anticodon is I, it can recognize ______ in the third base of the codon.

U, C, or A

True or False: the codons UUA and CUA (which both encode leucine) are read by the same tRNA because the 2nd and 3rd bases are the same and they code the same amino acid.

False; the codon UUA and CUA (which both encode leucine) are read by different tRNAs because codons that differ in either of their first two bases must be recognized by different tRNAs

True or False: the first base of an anticodon determines whether a particular tRNA molecule reads one, two, or three kinds of codons.

True

True or False: formation of a peptide bond between free amino acids is thermodynamically favourable.

False; formation of a peptide bond between free amino acids is thermodynamically unfavourable

Fill in the blanks: since the formation of a peptide bond between free amino acids is not thermodynamically favourable, the amino acid must first be activated in order for protein synthesis to begin. The activated intermediates in protein synthesis are _________. An _________ of tRNA is called an _________.

Amino acid esters, amino acid ester, aminoacyl tRNA (or charged tRNA)

Fill in the blanks: the activation of amino acids is catalyzed by ____________. The first step is the formation of an _________ from an amino acid and ATP. The next step is the transfer of the _________ group of _________ to a particular tRNA molecule to form __________, through an _______ linkage to 3’OH group of 3’ adenosine of the tRNA CCA arm.

Aminoacyl tRNA synthetases, aminoacyl adenylate (or aminoacyl-AMP), aminoacyl, aminoacyl-AMP, aminoacyl-tRNA, ester

What is the overall reaction of tRNA charging?

Activation by adenylation:

amino acid + ATP ←→ aminoacyl-AMP + PP_i

Transfer to tRNA:

aminoacyl-AMP + tRNA ←→ aminoacyl-tRNA + AMP

Overall reaction:

amino acid + tRNA + ATP + H₂O → aminoacyl-tRNA + AMP + 2P_i (consumes the equivalent of 2 ATP)

Multiple Choice: which class of enzymes does aminoacyl-tRNA synthetase belong to?

a) Oxidoreductase

b) Transferase

c) Hydrolase

d) Lyase

e) Isomerase

f) Ligase

g) Translocase

f)

True or False: aminoacyl-tRNA synthetase belongs to the transferase enzyme class.

False; aminoacyl-tRNA synthetase belongs to the ligase enzyme class because it joins two molecules together using the energy from ATP hydrolysis

Fill in the blank: each aminoacyl-tRNA synthetase is highly specific for a given amino acid. This level of specificity is achieved by each aminoacyl-tRNA synthetase taking advantage of ______________.

The properties of its amino acid substrate

Fill in the blanks: aminoacyl-tRNA synthetases have high specificity to their given amino acids. With threonyl-tRNA synthetase, its amino acid, threonine, is very similar to valine and serine. Threonyl-tRNA synthetase is able to prevent the coupling of these amino acids to tRNA^Thr by the __________ containing a ___ ion. This ion interacts with the _____ side chain of threonine, and not the side chains of valine or serine. Accidental coupling, especially with serine, does occasionally occur, but the synthetase has an __________ that accepts serine attached to tRNA^Thr and hydrolyzes the bond, removing serine.

Active site, Zn²⁺, OH, editing site

Fill in the blanks: with the threonyl-tRNA synthetase, valine cannot interact with the Zn²⁺ cofactor due to its _______ group in place of a(n) ______ group.

CH₃, OH

Fill in the blanks: with the threonyl-tRNA synthetase, serine can interact with the Zn²⁺ cofactor due to its _____ group and be added to the tRNA; however, it will be removed by _________ of the __________ linkage at the _________.

OH, hydrolysis, ester, editing site

Fill in the blanks: with the threonyl-tRNA synthetase, threonine cannot be removed at the _________ because it is ____________.

Editing site, too bulky to bind

Multiple Choice: which functional group prevents Thr-tRNA-Thr from fitting into the editing site of the corresponding aminoacyl-tRNA synthetase?

a) Methyl group

b) Hydroxyl group

c) Amino group

d) Carbonyl group

a)

Fill in the blanks: ribosomes are made of two subunits. In the E. coli ribosome, the small 30S subunit contains ___ proteins and a ___S rRNA. The large 50S subunit contains ___ proteins and two rRNA molecules, a ___S and a ___S species.

21, 16, 34, 23

Fill in the blank: the ribosome has ___ tRNA binding sites.

3

Fill in the blanks: the three rRNAs in the ribosome - ___S, ___S, and ___S - are critical for ribosomal architecture and function. These rRNAs are folded into complex structures with many short duplex regions.

5, 16, 23

Fill in the blanks: the tRNA binding sites in the 30S subunit of the ribosome are called the __________, the _________, and the _________.

A site (for aminoacyl), P site (for peptidyl), E site (for exit)

Fill in the blanks: