Exam 4: Lecture 1 Flashcards Preview

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Flashcards in Exam 4: Lecture 1 Deck (19):
1

Translation

-tasked with transferring mRNA transcript sequence into protein sequence

2

Ribosome Subunits

-consists of small subunit and large subuinit
-each subunit consists of multiple different proteins
-both associated with non-coding RNAs (ncRNA)

3

ncRNA

-aids in stabilizing interactions with mRNA transcript

4

Translation Brief Process

-mRNA transcript bound by small ribosomal subunit
-initiator tRNA bound to modified Methionine (fMet) brought into contact with first AUG codon of coding sequence
-large ribosomal subunit added to complex
-complete ribosome reads transcript 5' to 3' and generates appropriate protein sequence
-process repeated till mRNA transcript is degraded

5

Polysomes/Polyribosomes

-multiple ribosomes that bind each mRNA transcript
-increases amount of protein produced prior to degradation because multiple ribosomes are simultaneously translating mRNA

6

Increasing Protein Production

-mRNA transcript organized into circular structure
-5' UTR and 5' CAP structure bound initiation factors that interact with Poly-A Binding Protein which is bound to 3' Poly-A tail
-circular conformation protects ends from degradation and can be translated by multiple ribosomes for longer period of time than linear mRNA (prefer to degrade single stranded DNA)
-eventually falls apart

7

Ribosome Composition

-small and large subunit
-subunits composed of many individual proteins

8

Centrifugation

-used in early attempts to understand ribosome structure
-intact ribosomes placed in sucrose solution and spun at high velocities
-size of protein complex directly proportional to sedimentation velocity
-i.e. intact ribosome travels faster through gradient than small or large individual subunits
-likewise large subunit sediments at higher velocity than smaller
-after isolated, further disrupted to yield individual proteins sequenced by Fred Sanger's methods

9

A, P, and E sites

-three channels for tRNA binding within ribosome
-initiator tRNA recruited to P site where it interacts with first AUG within coding region of mRNA transcript
-once initiator tRNA and AUG codon intact with P site, large subunit recruited

10

Alignment

-Aligning first codon with P site automatically ensures second codon aligned with A site
-second tRNA enters ribosome and interacts with transcript via A site
-peptide bond forms between first two amino acids then ribosome moves to next codon.
-moves initiator tRNA into E pocket where it's ejected
-second tRNA moves into P pocket leaving empty A pocket
-each subsequent tRNA enters ribosome through A site to interact with mRNA

11

Translation Initiation Sequences

-ribosome must be recruited to 5' end of mRNA transcript
-prokaryotes: Shine-Dalgarno sequence located 3-9 bases upstream first AUG
-eukaryotes: Kozak sequence encompasses few bases upstream and downstream AUG

12

Binding to Initiation Sequences

-after binding to sequences AUG placed in P pocket
-non-coding ribosomal RNAs associated with small ribosomal subunit important in interacting with mRNA and for ensuring first AUG aligned with P pocket

13

Initiation of Prokaryotic Translation: Blocks on Sites

-initiation factors bind to E and A sites to block initiator tRNA from entering

14

Initiation of Prokaryotic Translation

-steps after blocking
-mRNA and initiator RNA recruited to small ribosomal subunit
-contact between ribosome and mRNA mediated by RNA:RNA interactions
-16S ribosomal RNA base pairs with Shine-Dalgarno sequence in mRNA
-5-9 base sequence that allows just enough space for AUG to be in P pocket which ensures AUG's position
-large ribosomal subunit recruited to make complete ribosomal unit
-anticodon of initiator tRNA makes contact with AUG codon in P pocket begins elongation
-IF's (1 and 3) removed from A and E pockets

15

Length of Sequence for Alignment

-too long, AUG won't line up in P pocket (may be in A pocket or completely out of ribosomal subunit)
-too short also causes problems

16

Differences in Eukaryotic Translation

-initiator tRNA recruited to small ribosomal subunit independently of mRNA transcript
-recruitment of transcript does not ensure alignment of initiator

17

Similarities between Prokaryotic and Eukaryotic Translation

-number of initation factors required to displace large ribosomal subunit and to block E and A channels
-leaves P channel only one open for tRNA

18

Translation Elongation (study example in notes)

-subsequent tRNAs (after initiator) enter A pocket
-second codon enters and codes for amino acid
-tRNA attached to that protein recruited to A pocket where anticodon pairs with the codon
-Met transferred to tRNA containing the amino acid coded for and a peptide bond will form between the Met and other amino acid
-ribosome moves over slightly moving AUG codon into E pocket and the subsequent is in P pocket and third is in A pocket
-empty tRNA in E pocket expelled and A pocket is filled by tRNA that base pairs with codon
-process repeated again and again until ribosome reaches stop codon
-then dissociates from mRNA and nascent protein is released

19

Eukaryotic Translation

-ready to interact with mRNA transcript when pre-initiation complex has formed
-5' end of mRNA bound by IFs that interact with IFs bound to small ribosomal subunit
-recruitment of mRNA to small ribosomal subunit does not result in alignment of initiator AUG in P pocket (difference)
-small ribosomal subunit moves along mRNA until it finds AUG which is recognized when anticodon of tRNA bases pairs with initiator codon
-initiator codon:anticodon binding stabilized, large ribosomal subunit recruited to make complete ribosome
-IF blocks expelled from E and A pockets and elongation begins