1
Q
Transcription and translation are the mechanisms by which cells ___________________
A
Transcription and translation are the mechanisms by which cells express their genetic information
2
Q
What TWO general factors contribute to cells being able to synthesize a large amount of protein from a single gene?
A
1) The ability to generate many identical RNA copies from the same gene (multiple rounds of transcription)
2) The ability for each mRNA to direct the synthesis of multiple identical copies of a given protein (multiple rounds of translation)
3
Q
Explain the consequences of differing transcription efficiency
A
By allowing different genes to be transcribed/translated with different efficiencies, a cell can make a ton of some proteins and a tiny amount of other proteins
4
Q
Transcription and translation efficiency contribute to determining ______________
A
Transcription and translation efficiency contribute to determining protein abundance in a cell
5
Q
What important features does the single stranded nature of RNA give to the molecule?
A
Being single stranded allows an RNA chain to fold up into complex 3D SHAPES by complementary base pairing between regions within the same RNA
6
Q
Why is RNA 3D structure important?
A
3D structures of RNA allows some RNAs to have very precise structural and catalytic functions!
7
Q
Define transcription
A
The copying of one strand of DNA into a complementary RNA sequence by an RNA polymerase
8
Q
Explain the broad, overall process of transcription (3):
A
1) Opening + unwinding of a select portion of DNA
2) Use of one DNA strand as a template for RNA elongation
3) “Progressive release” of transcript
9
Q
What is meant by progressive release of a transcript?
A
The elongating RNA strand DOES NOT remain hydrogen-bonded to the DNA template strand!
–> There is only a small section of around 10 NTs of RNA that remain bound to the DNA template strand
–> In the RNA behind this region, the RNA bound to the DNA template is displaced by the DNA’s original partner == reforms the DNA helix!
10
Q
Define RNA Polymerases
A
Enzymes that catalyze the synthesis of an RNA molecule on a DNA template using ribonucleoside triphosphates (rNTPs) as substrates
11
Q
What do RNA polymerases catalyze?
A
The formation of PDE bonds that link NTs together in an RNA chain
12
Q
How is the DNA template unwound for transcription?
A
By RNA polymerase!
–> As RNA pol. moves stepwise along a DNA template, it unwinds the DNA helix just ahead of its polymerization active site
13
Q
In what direction is RNA made?
In what direction is the DNA read?
A
RNA is elongated 5’ —> 3’
DNA is read by RNA Pol. 3’ —> 5’
14
Q
Explain how RNA and DNA interact during transcription:
A
1) In the RNA pol. active site, there is a short segment of RNA-DNA heteroduplex
== A short region of DNA-RNA helix that is formed transiently
2) After a given synthesized RNA portion is elongated out of the active site, the DNA template is separated from the transcript
3) The 5’ end of the growing RNA strand begins extending OUT of RNA pol. and the double helix of the DNA is restored
15
Q
What are the implications of the DNA template and transcript being separated quickly?
A
This allows MULTIPLE RNA COPIES to be made from a gene in a short period of time!
== An RNA pol. can start UPSTREAM (behind) an RNA pol. transcribing a segment of the gene downstream!
(additional RNA molecules can begin synthesizing before the previous RNAs are completed)
16
Q
List some differences between RNA polymerase + DNA polymerase (3):
A
RNA pol…
1) Does NOT require a primer! (de novo synthesis)
2) Has no 3’ –> 5’ proofreading ability
3) Is processive
17
Q
Why does RNA polymerase NOT need a primer?
A
Because there is really no need for 3’ –> 5’ proofreading capability as an error in transcription is not very serious as RNA does not store + transmit genetic information
–> RNA exists transiently, so why waste the energy on trying to make sure it’s highly correct?
= If no proofreading is needed, then the requirement of a proper primer (which allows for this kind of proofreading) is not needed!
18
Q
Explain what is meant by “RNA Polymerases are processive”:
(AND how does this relate to transcription?)
A
RNA polymerases are processive == the SAME RNA polymerase that begins an RNA molecule must FINISH that molecule without dissociating from the template!
== An RNA transcript CANNOT be made by RNA polymerase alternating between adding NTs, dissociating, reassociating, and repeating!)
If RNA polymerase falls off during transcription, it must start ALL OVER AGAIN
19
Q
List some important types of RNA:
A
1) mRNA (messenger RNA)
2) ncRNA (noncoding RNA)
–> rRNA (ribosomal RNA)
–> tRNA (transfer RNA)
–> snRNA (small nuclear RNA)
20
Q
Define mRNAs
A
mRNA = messenger RNAs
–> RNA molecules that specify the AA sequence of a protein and undergo translation to produce an encoded protein
–> mRNAs are RNAs transcribed from CODING GENES!
21
Q
Define ncRNAs
A
ncRNA = noncoding RNAs
–> RNAs that are the final gene product of noncoding genes
–> These RNAs do not encode for protein
–> A wide array of ncRNAs exist that serve enzymatic, structural , and regulatory roles in various cell processes (Ex: tRNA, rRNA, snRNA…)
22
Q
Define tRNAs
A
tRNA = transfer RNAs
–> A form of ncRNAs that function as adaptors that select AAs + hold them in place on a ribosome for incorporation into proteins (essential for translation)
23
Q
Define rRNAs
A
rRNA = ribosomal RNAs
–> ncRNAs that form the CORE of ribosomes; carry out the critical enzymatic activites of the ribosome!
24
Q
Degine snRNAs
A
snRNA = small nucleolar RNAs
–> ncRNAs that direct the splicing of pre-mRNA to form mature mRNA (make up the spliceosome)
25
What is a *transcript*?
The RNA chain made by transcription
26
What is a *transcriptional unit*?
A segment of DNA that gets transcribed
27
Explain how eukaryotes + bacteria differ in the contents of their transcriptional units:
**Eukaryotes:**
ONE transcriptional unit typically contains the info for ONE gene
== Encodes for a single RNA/protein product
**Bacteria:**
One transcriptional unit may contain SEVERAL adjacent genes
== Transcript will carry the info for a # of products!
28
What is the starting point for RNA polymerase?
PROMOTER
29
What is a *promoter*?
NT sequence in DNA to which RNA polymerase (+ other factors) bind to begin transcription!
30
Explain the process of transcription initiation in BACTERIA:
1) RNA polymerase binds to **σ-factor (sigma)**, creating the RNA polymerase *holoenzyme*
2) The holoenzyme then slides along the DNA "looking" for a promoter sequence
3) When a promoter sequence is identified, the holoenzyme binds tightly
4) *Abortive initiation begins:* A period of inefficient transcription as the complex is "stuck" to the promoter + produces irrelevant, "junk" transcripts
5) The tightly bound holoenzyme "breaks free" of its interaction with the promoter, *moving downstream of the promoter + releasing σ-factor (sigma)*
6) RNA pol. shifts to elongation mode + begins actual transcription
31
What is *σ-factor (sigma)*?
A **bacterial transcription factor (protein)** that binds RNA polymerase to form the holoenzyme
== Enables RNA polymerase to bind specific gene promoters! (by recognizing a promoter, binding to it, and bringing the attached RNA pol. to it)
32
If RNA polymerase dissociates prematurely, what happens?
If RNA polymerase dissociates prematurely, before finishing the transcript it is elongating...
It must **START OVER** again at the promoter!
33
Explain the 3 kinds of RNA polymerases in eukaryotes + their functions:
**RNA Pol. I** == Transcribes MOST rRNA genes (all but one)
**RNA Pol. II** == Transcribes ALL protein-coding genes (mRNA) + many different ncRNAs (like snRNAs)
**RNA Pol. III** == Transcribes tRNA genes + ONE rRNA gene
34
Which RNA polymerase generates mRNA?
RNA Polymerase II
35
Which RNA polymerase generates tRNA?
RNA Polymerase III
36
Which RNA polymerase generates snRNA?
RNA polymerase II
37
Which RNA polymerase generates rRNA?
Primarily, *RNA Polymerase I* (3 rRNAs)
Also, *RNA Polymerase III* (1 rRNA)
38
What are *general transcription factors*?
General TFs are proteins that bind to key regions in ALL gene promoters to help RNA polymerase bind, activate, and begin initiation at the correct site
(called "general" because these TFs are needed for the initiation of ALL genes!)
39
How do general + specific TFs differ?
*General TFs* == bind to ALL promoters, triggering *base-level* transcription to occur
*Specific TFs* == bind to regulatory sequences such as repressors or enhancers to increase fine-tune the level of transcription
40
List reasons why general TFs are needed for RNA pol. II transcription initiation: (3)
1) Help position RNA polymerase II correctly at a promoter
2) Aid in pulling apart the 2 DNA strands to allow transcription to begin
3) Help release RNA polymerase from the promoter to start its elongation
41
What are *TFII* proteins?
The general transcription factors for RNA Polymerase II
42
What consensus sequence is critical in the promoter?
**TATA BOX**
43
What is the *TATA Box*?
A consensus sequence of alternating T/A in the promoter of most eukaryotic genes that binds a general TF, thereby specifying the position at which transcription is initiated
44
Where is the TATA box usually found?
The TATA box is usually 30 NTs *UPSTREAM* of the transcription start site (AUG)
45
Explain the process of transcription initiation:
1) The first general TFII binds to the TATA box of a promoter causing a large distortion in the DNA sequence, facilitating further initiation complex assembly
2) Additional TFIIs bind to the promoter + each other
3) RNA polymerase binds to the promoter + associates with the bound TFIIs
== **Initiation Complex Formed**
4) One bound TFII hydrolyzes ATP to pull apart the 2 DNA strands to allow RNA polymerase to access the DNA template
5) A TFII phosphorylates the C-terminal tail of RNA polymerase causing the RNA pol. to release from the general TFs + promoter
== RNA pol. tightens hold on DNA + allows for association with elongation factors, transcription begins!
46
What is the purpose of phosphorylating the C-terminal tail of RNA polymerase?
**1) To initiate the enzyme's release from the promoter so it can begin transcription**
**2) To create "docking sites" for RNA processing machinery to load onto**; allowing the processing machinery to be properly oriented to process transcripts as they emerge from the enzyme
47
In addition to general TFs, what other factors are required for transcription initiation? (3)
**1) Transcriptional Activators**
**2) Mediators**
**3) Chromatin Modifying Enzymes**
(chromatin remodeling complexes, histone modifying enzymes)
48
What are *transcriptional activators*?
--> Regulatory proteins that bind *enhancers* that help attract general TFs + RNA pol. to the promoter
49
What are *mediators*?
Large protein complexes that allow transcriptional activators to communicate properly with RNA Pol. II + TFs!
(Also helps organize the initiation complex to position machinery properly for RNA Pol. II C-terminal tail)
50
Why are chromatin modifying enzymes needed for transcription initiation?
Needed to increase access to DNA in chromatin to facillitate the assembly of the initiation complex on DNA
51
What is an *enhancer*?
A short DNA sequence that acts as a binding site for TFS, specifically *transcription activators* == significantly increases transcriptional activity!
52
What is the "problem" with enhancers?
And what is the "solution" to this?
**"Problem" ==** Enhancer sequences are usually very far from the genes they regulate
**"Solution" =** *"Looping Mechanism"*: Enhancers function by looping or folding over to bring their sequence close to the region of DNA they regulate!
--> Once in close proximity, the enhancer + bound activator facilitate the assembly of the initiation complex at a promoter
53
What is the main role of mediators?
To coordinate the assembly of all the proteins at the promoter to help transcription begin
54
What are *elongation factors (EFs)*?
EFs are proteins that decrease the likelihood that RNA polymerase will dissociate before reaching the end of a gene
(increase processivity)
55
When do EFs typically associate with RNA polymerase?
EFs typically associate with RNA pol. quickly after the initiation factors dissociate + initiation has been achieved
56
What 2 main factors do EFs help with?
1) EFs help RNA polymerase move through nucleosomes
2) EFs help RNA polymerase continue elongation through various sequences (some that are easy to transcribe and others that are more difficult)
57
How do EFs function in relation to RNA polymerase continuing through nucleosomes?
Certain EFs associated with RNA polymerase will form a "wedge" at the front of RNA pol. == **pries DNA away from its histone core as RNA polymerase moves forward**!
58
What are the overall steps to go from DNA to protein in bacteria vs. eukaryotes:
**BACTERIA:**
1) Transcription
2) Translation
**EUKARYOTES:**
1) Transcription
2) RNA Processing (5' Capping, Splicing, 3' Polyadenylation)
3) Nuclear Export
4) Translation
59
How do eukaryotes + bacteria differ in terms of WHERE transcription + translation take place?
BACTERIA == All occurs in ONE compartment (cytosol)
EUKARYOTES == Compartmentalized; Transcription in the nucleus + Translation in the cytosol
60
What is the importance of 5'-capping and 3'-polyadenylation?
These two modifications allow a cell to assess whether both ends of an mRNA are present (i.e. transcription completed successfully) before exporting the mRNA from the nucleus + translating it!
61
Explain the sequence of RNA processing events as RNA polymerase functions
1) 5'-Capping proteins bind to the phosphorylated residues on the C-terminal tail of RNA pol.
2) As the 5' end of the nascent mRNA emerges from the RNA polymerase, the nearby docked 5'-Capping proteins "HOP" onto the 5' end of the nascent mRNA + the cap is formed
3) Splicing proteins + machinery dock on the phosphorylated residues on the C-terminal tail of RNA pol.
4) Splicing machinery hops onto the emerging mRNA at different points to begin splicing
5) Poly-adenylation proteins dock on the C-terminal tail
6) When the end of the transcript begins to emerge, the poly-A proteins add the Poly-A tail to the transcript!
62
What is the 5'-Cap made of?
A modified guanine NT == **7-methylguanosine**
63
Explain the chemical process of 5'-capping:
1) The triphosphate grp at the 5' end of the emerging transcript gets cleaved to release the GAMMA-phosphate
2) GTP is hydrolyzed to release PPi and attach GMP to the 5' end of the transcript (specifically creates a bond between the BETA-phosphate of the transcript and the phosphate of the GMP)
3) The guanosine in the added GMP is methylated == forms the modified guanine NT cap!
64
Explain the importance of 5' capping: (3)
1) The 5'-cap on mRNA serves as a "landmark" to cellular machinery that distinguishes mRNA frm other RNAs in the cell!
2) The 5'-cap binds to CBP (cap binding protein) which is needed for nuclear export
3) Critical to translation, ensuring it begins at the correct site
65
What is *RNA Splicing*?
A process in *eukaryotic* nuclei that involves the excision of intron sequences from RNA transcripts leading to mRNA formation
66
Why is splicing needed in eukaryotes but NOT in bacteria?
Splicing is needed in eukaryotes because **genes contain alternating introns + exons which BOTH get transcribed**
== The introns need to be removed from the initial transcript
Splicing is NOT needed in bacteria because they lack introns! == no splicing out of introns are needed as there are no introns that end up in the transcript
67
Explain the process of one splicing event:
1) The 2' OH of a specific adenine in an intron sequence attacks the 5'-splice site (junction between previous exon + the intron)
--> This attack cuts the PDE bond between the 5' end of the intron + the 3' end of the previous exon AND *forms a new PDE between the internal intron Adenine and the 5' end of the intron*
2) The released/free 3'OH end of the previous exon (at the 5' splice site) attacks the PDE at the 3' splice site (junction between the intron and the next exon)
--> Breaks the PDE bond between the 3' end of the intron + the 5' end of the next exon AND *forms a new PDE between the two exons, connecting them together*
3) The intron is released as a **LARIAT** + gets discarded
68
What is *alternative splicing*?
A splicing process in which EXONS can be spliced in different ways to create transcripts with different combinations of exons
(multiple possible mRNAs from 1 pre-mRNA)
69
Why is alternative splicing important?
Allows one gene to be able to produce a set of different but related proteins through the creation of slightly differing mRNAs!
70
What 3 portions of the pre-mRNA does splicing machinery need to recognize in order for splicing to occur?
HOW are these sites recognized?
1) 5' Splice Site
2) 3' Splice Site
3) Branch point in the intron (ADENINE) that forms the base of the excised lariat
--> *each of these sites has a CONSENSUS SEQUENCE that splicing machinery recognizes!*
71
Explain what machinery is needed for splicing:
**snRNAs** carry out the key processes of splicing
--> These snRNAs complex with around 7 proteins forming **snRNPs** (small nucleolar ribonucleotide proteins) which make up the core of the *spliceosome*
72
What is the *spliceosome*?
A large assembly of RNAs + proteins that perform pre-mRNA splicing whose core is primarily made up of snRNPs
73
What snRNP/s functions first? What does it/they do?
**U1**and **U2**
--> U1 binds to the sequence at the 5' splice site on pre-mRNA
--> U2 binds to the branch point sequence (ADENINE) on pre-mRNA
74
What snRNPs are responsible for the actual catalysis of splicing?
U2 and U6!
75
Explain the process of splicing in terms of snRNP action:
1) U1 binds to the 5' splice site
2) U2 binds to the branch point sequence (adenine)
3) U6 displaces U2 at the 5' branch point == triggers the U2 + U6 snRNPs to move closer to each other, creating a loop in the pre-mRNA containing the intron + forming the active site for splicing
4) U2 + U6 catalyze the middle-A attack of the 5' splice site + ultimate release of the intron as a lariat
76
How are snRNPs able to recognize splice-site sequences?
Through complementary base pairing between the RNA in the snRNPs and the sequences in the pre-mRNA
77
What is the difference between bacteria + eukaryotes in terms of how transcription terminates?
**Bacteria** == Contain *termination sequences* that cause RNA polymerase do dissociate when the enzyme reaches them
**Eukaryotes** == DO NOT HAVE termination sequences! So, RNA polymerase often continues transcription beyond the final codon and 3'-polyadenylation process corrects this
78
Explain the process of 3'-polyadenylation + transcription termination:
1) 3' End processing proteins bind to their recognition sequences near the ACTUAL end of the transcript as RNA polymerase continues improper elongation
2) The proteins direct the cleavage of the pre-mRNA, releasing the PROPER 3' end from RNA polymerase! **AND** the recruitment of all these proteins begins to slow RNA polymerase down
3) Poly-A Polymerase then adds ~200 adenine NTs to the proper 3' end of the pre-mRNA produced by the cleavage step
4) Once the 3' end of the pre-mRNA is cleaved, a free 5' end is created on the RNA still coming out of RNA polymerase --> This 5' end LACKS A CAP and thus gets degraded
== Once degradation catches up to RNA polymerase, the enzyme STOPS + dissciates!
79
When can we actually call a transcript "mRNA"?
AFTER all the processing steps have been completed! (splicing, capping, polyadenylation)
80
How are mRNAs distinguished from other RNAs for their selective export from the nucleus?
**The acquisition of certain proteins that signal the completion of the 3 main mRNA processing events!**
*1) Association of CBP (cap binding protein)* == signals completion of the 5'-capping process
*2) Association of exon junction complexes* == signals completion of splicing
*3) Association of Poly-A Binding Proteins* == signals completion of poly-adenylation
81
What enzyme carries out polyadenylation?
Does this enzyme require a template? Why or why not?
**Poly-A Polymerase**
--> This enzyme does NOT require a template as it just adds the same NT over and over again (why the Poly-A tail is not encoded in the genome)
82
Only when the _______ present on an mRNA molecule __________ signify that __________ was successfully completed is the mRNA exported from the nucleus
Only when the **proteins** present on an mRNA molecule **COLLECTIVELY** signify that **processing** was successfully completed is the mRNA exported from the nucleus
83
What happens to improperly processed mRNAs?
They remain in the nucleus (don't get exported) and get degraded
84
How are mRNAs exported out of the nucleus?
Through the **nuclear pore complexes** in an **ACTIVE TRANSPORT** process requiring ATP
85
What targets mRNAs to the nuclear pore complexes?
The association of an mRNA with a **nuclear pore receptor** which is added during poly-adenylation
86
Proteins acquired in the nucleus that remain associated with an mRNA in the cytosol determine... (3)
1) The stability of mRNA in the cytosol
2) The efficiency with which an mRNA is translated
3) An mRNA's ultimate destination in a cell
87
The cell requires A LOT of rRNA, how is the cell able to keep up with demands?
By containing **MULTIPLE COPIES** of rRNA genes!!
88
What does the nucleolus contain in terms of ribosome production?
1) rRNA genes
2) Pre-rRNAs
3) Mature rRNAs
4) rRNA processing enzymes
5) snoRNPs
6) Ribosomal proteins
7) Partly assembled ribosomes
89
The nucleolus is the site for...
the synthesis + processing of rRNAs and the assembly of ribosomes
90
The size of the nucleolus reflects...
The # of ribosomes a cell is producing
91
Define *translation*
Process by which the sequence of NTs in an mRNA directs the incorporation of AAs into protein (occurs in a ribosome)
92
What is the *"genetic code"*?
The set of "rules" specifying the correspondence between codons in DNA/RNA and AAs in proteins
93
What is a *codon*?
A sequence of 3 NTs in DNA/mRNA that encodes for a given AA
94
What is meant by *"redundant code"?*
Several codons encode for the same AA!
(often the redundant codons have the same first 2 NTs and differ only in the third NT)
95
What is the START codon?
AUG
96
What are the STOP codons?
3 of them:
1) UAG
2) UAA
3) UGA
97
Any RNA sequence can be translated in any one of 3 __________
Any RNA sequence can be translated in any one of **3 Reading Frames**
98
What are *reading frames*?
The "frame" in which NTs are read in sets of 3 to encode proteins
99
What determines the reading frame?
Where the decoding/translation process begins on an mRNA
100
Do codons directly bind to the AA they encode?
NO --> the codon sequence binds to the ANTICODON sequence of a tRNA carrying an AA that corresponds to the AA encoded by the codon
101
What is the 3D structure of tRNAs?
"Cloverleaf" structure with 4 short regions of dsRNA
102
What are the 2 critical regions of tRNAS?
WHY?
2 critical single stranded regions of tRNAs:
**1) Single stranded 3' end**
== Site where the proper AA binds to the tRNA molecule
**2) Anticodon**
== pairs to a codon on the mRNA to direct the addition of its AA to the growing polypeptide chain
103
What is an *anticodon*?
A sequence of 3 NTs in a tRNA that pairs with the complementary CODOn in an mRNA molecule
104
Explain the *specificity* of tRNAs:
Some tRNAs are able to bind more than one codon!
== Can bind the other redundant codons encoding for the same AA as the tRNA allows for a "wobble" (mismatch) of the last NT in a codon sequence
**All the codons a tRNA can bind are those that encode the SAME AA!**
105
What enzyme couples tRNAs to their proper AA?
*Aminoacyl-tRNA Synthetases*
106
What is an *aminoacyl tRNA*?
A tRNA with its proper AA covalently attached to its 3' end (= a "charged" tRNA)
107
Explain the process of *tRNA "charging"*
1) ATP is hydrolyzed to release PPi + add AMP to the carboxyl end of an AA == activates the AA!
2) The 3'OH of the proper tRNA attakcs the carbonyl carbon that is adenylated == triggers release of AMP + forms an *ester linkage* betweem the AA and the tRNA
108
Why is the ester linkage between an AA and tRNA in an aminoacyl-tRNA important?
Because it is a high energy bond whose breakage later in protein synthesis will drive the addition of AAs to a polypeptide chain
109
What is the fundamental reaction of protein synthesis?
The formation of a peptide bond between the carboxylic acid end of a growing polypeptide chain and the amino end of an incoming AA
110
What is the direction of protein synthesis?
Proteins are made from their **N-terminus ---> C-terminus**
111
What is a *peptidyl tRNA*?
A tRNA with its 3' OH attached to the carboxyl end of a growing polypeptide chain
112
In translation, each AA addition disrupts the __________ but immediately ________ with an __________on the most recently added AA
In translation, each AA addition disrupts the **high energy ester linkage** but immediately **replaces it** with an **identical linkage** on the most recently added AA
113
What is *polymer-end activation mechanism*?
Each AA added to a polypeptide chain carries with it the energy for the addition of the NEXT AA (rather than the energy for its own addition)
114
Explain the structure of both subunits of a ribosome:
Large Subunit == 3 rRNAs + many proteins
Small Subunit == 1 rRNA + many proteins
115
The small ribosomal subunit provides the ______ on which the tRNAs are ___________ to the mRNA codons
The small ribosomal subunit provides the **framework** on which the tRNAs are **accurately paired** to the mRNA codons
116
When ribosomes are NOT actively synthesizing proteins...
The 2 subunits are NOT assembled together!
--> Ribosomal subunits only come together ON an mRNA!
117
Explain the binding sites in a ribosome:
4 of them:
1) **A-SITE** == Aminoacyl-tRNA site; where new charged tRNA enters the ribosome
2) **P-SITE** == Peptidyl-tRNA site; where the tRNA carrying the polypeptide chain sits to wait for transfer to the next Aminoacyl-tRNA
3) **E-SITE** == Exit site; where uncharged tRNAs leave the ribosome
4) **mRNA binding site** == in the small subunit, where mRNA binds to align with the A, P, and E sites
118
Explain the 4 major steps of translation:
**1) tRNA Binding** == A newly charged tRNA enters and binds to the free A-site in the ribosome
**2) Peptide Bond Formation** == The N-terminal of the AA on the tRNA in the A site attacks the ester linkage of the peptidyl-tRNA == cleaves the chain from the tRNA in the P-site and connects the growing chain to the AA held by the tRNA in the A site!
**3) Large Subunit Translocation** == The large subunit moves relative to the mRNA held by the small subunit by 3 NTs --> Shifts the A, P, and E sites of the large ribosome over one codon
== the uncharged tRNA is now in the E site and the new peptidyl-tRNA is now in the P-site
**4) Small Subunit Translocation** == A series of conformational changes causes the small subunit to move 3 NTs to catch up to the large subunit
== in this process the uncharged tRNA is released from the E-site and now everything is reset to begin again!
119
What is the ratio of RNA to protein in ribosomes?
2/3 RNA + 1/3 protein
120
In ribosomes rRNAs are responsible for... (3)
1) The ribosome's overall structure
2) The ribosome's ability to position tRNAs on the mRNA
3) The ribosome's catalytic activity in forming peptide bonds
121
What are *elongation factors* in translation?
Proteins that enter + exit the ribosome during translation that hydrolyze GTP to induce changes that are coupled to transitions between different ribosomal states to increase the speed of protein synthesis!
122
What are the main functions of ribosomal proteins?
To stabilize the rRNA core while permitting changes in the rRNA conformation that are necessary for catalysis
123
Where are ribosomal proteins made and how do they meet up with rRNAs for assembly?
Ribosomal proteins are made in the cytosol and then imported into the nucleus for assembly in the nucleolus
--> *chaperones* escort the largely unfolded forms of these proteins into the nucleus
124
What are ribozymes?
RNAs that possess catalytic activity (ribosome, spliceosome)
125
What is one determinant of the rate at which any given protein will be synthesized?
The efficiency of translation initiation
126
What is an *Initiator tRNA*?
A specialized tRNA that initiates translation --> Always carries methionine + recognizes the AUG START codon
127
How is tRNAi different from tRNA(met)?
tRNAi has specialized sequences that are specially recognized by initiation factors unlike typically methionine tRNAs!
128
Explain the process of translation initiation:
1) tRNAi + elongation initiation factors (eIFs) bind to the P-SITE of the small subunit of a ribosome
2) The small subunit binds the 5' end of an mRNA which is recognized by an eIF binding to the 5' cap of the molecule
3) The small subunits moves forward along the mRNA (5' --> 3') "searching" for the first START (AUG) codon!
4) Once the first AUG codon is found, the small subunit stops, eIFs dissociate and the large subunit is able to bind + assemble the complete ribosome!
129
What influences the accuracy of the "scanning" process carried out in translation initiation?
The NTs surrounding the first AUG sequence on the mRNA!
--> The machinery is used to a specific sequence around the first AUG; if the first AUG has neighboring NTs that differ from normal, the small subunit may PASS that AUG and continue on until it finds the next one!
130
Almost all proteins have WHAT AA at their N-terminal *initially*? WHY?
**Methionine**:Because the tRNAi carries methionine!
131
How does bacterial translation initiation work? Why is it different from ours?
**Bacterial mRNAs do NOT have a 5'-cap! == no signal of where to start scanning for the start codon!**
--> Instead, ribosomes scan the mRNA for *shine-dalgarno sequences* which serve as ribosomal binding sites that are a few NTs upstream of the first start codon!
132
What can bacterial ribosomes do that we cannot in translation?
Due to the Shine-Dalgarno Sequence-Mediated translation initiation, bacteria can initiate translation at an INTERNAL position of an mRNA (don't have to start at the end like we do)
133
What is special about the STOP codons?
They are not recognized by any tRNA + therefore do not specify the addition of any AA!
134
What are *release factors*?
Proteins that bind to the A-SITE of a ribosome when a STOP-codon is presented!
== **Force the ribosome's catalytic machinery to add WATER instead of an AA to the peptidyl-tRNA!**
135
Explain the process of translation termination:
1) A stop codon is presented in the mRNA at the A-site
2) A release factor enters the A-site and binds
3) Binding of the release factor triggers the ribosome to catalyze the addition of water to the C-terminal of the growing polypeptide chain == the ester linkage to the peptidyl-tRNA is cleaved and the COOH end of the protein is created!
4) The complete protein releases from the ribosome through a water-filled exit tunnel
5) The ribosome disassembles
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What is a *polysome*?
A large cytoplasmic assembly made up of several ribosomes spaced apart along a single mRNA
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Why is a polysome important?
It allows mltiple initiations of translation to occur simultaneously on an mRNA which allows the cell to make many more proteins!
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How does a polysome work?
As soon as a ribosome has translated enough of the mRNA seq to move out of the way of the 5' start region, the 5' end is threaded into ANOTHER ribosome to carry out translation simultaneously!
BIO 310
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