mod 4 cont'd: genome 2,3,4

what is the folding of RNA?

complex 3D structures

stem + loop(, G-U stabilizes end of hairpin), hairpin structures

what does folding enable in RNA structure?

catalytic and structural roles.

what are all the types of RNA?

mRNA, tRNA, rRNA, snRNA, snoRNA, miRNA, siRNA, IncRNA

what is RNA polymerase?

synthesis of complementary RNA, reads 3’→5’ but synthesies in 5’→3’

is primer required for initiation of RNA synthesis?

no primers are required

what are promoters?

DNA sequences upstream of a genes transcription start site +1

the TATA box is the recognition site. serves as the start codon

what does the TATA box define?

transcription start site and direction

what are the three steps of transcription?

1. initiation: RNA polymerase binds to promter, unwinds DNA, begins RNA synthesis

2. elongation: RNA polymerase moves along DNA synthesizing RNA

3. termination: RNA polymerase encounters trancsription stop signal in DNA

how does bacterial promoters start transcription?

-35 element

• upstream from +1; recognition and binding site for sigma factor ( enables promoter recognition and transcription initiation)

-10 element

• A-T rich region that melts easily to form open complex to get transcribed.

how does elongation occur in prokaryotic transcription? (bacterial)

after 10nts sigma factor dissasociates and elongation complex forms

RNA polymerase moves along template, synthesizing RNA 5’→3’

what are the three different RNA polymerases?

pol. 1 most rRNA

pol.2 mRNA

pol.3 tRNA

multi subunits, RNA transcript exits via channel

what does the eukaryotic transcription process require?

general transcription factros

always needed to initiate

what is the preinitiation complex?

GTF assemble with pol.II to form the preinitiation complex (PIC)

corelelments contain the TATA box, BRE, initiator

how does initiation start from promoters?

1. TFIID

• TBP( TATA binding protein)subunit binds to TATA box

• TBP associated factors (TAFs) needed for binding to occur

2. recruitment of GTF’s

• TFIIB positions RNAPII

• TFIIA

3. recruitment of RNAPII-TFIIF

• TFIIF stabilizes interaction between RNAPII and TBP & TFIIB

4. recruitment of additional GTF’s

• TFIIH kinase and helicase activity

• TFIIE

PIC is now formed and RNAPII is active

where are mRNA’s processed?

in the nucleus

transported out to the cytosol for translation to take place

how is nucler transport of mRNA’s facilitated?

through nuclear pores

before processing can occur RNA processing steps must occur first

(before= pre-mRNA)

what are the steps of RNA processing?

1. capping- modification of 5’ end and takes place after 25 nts. includes guanine + methyl groups

2. splicing- removes introns, joins exons

3. polyadenylation- added poly A tail at 3’ end for degradation during transport

what are the structures of mRNA’s?

continuous coding sequence

5’ methylated cap

3’ poly-A tail

what are split genes?

difference in in size between hetereogenous nuclear DNA

intervening sequences = intron (noncoding)

expressed sequences = exon (coding)

why does RNA splicing increase protein diversity?

different combinations of exons joined making unique mRNA’s

→ enables tissue specific protein expression

what is a spliceosome?

removes introns, joins exons

what is transcriptional control regulated by?

1. general TF’s

• bind to core promoter, recruits RNA pol. II

2. sequence specific TF’s

• bind to various regulatory sites of particular genes

what are transcriptional activators?

stimulate transcription

what are transcriptional repressors?

inhibit transcription

what two domains do TF’s contain in structure?

DNA binding domain: region specific DNA motifs

activation domain: interacts w/ other proteins to modulate transcription

they form dimers for stability and specificity

what kind of roles do TF’s play in gene expression?

• extent of transcription depends on combination of TF’s bound to upstream regulating elements

• combinations can differ in cells of different type tissue and stages

• 5-10% of genes encode TF’s

• **combinatorial control of transcription: Oct4

what is combinatorial control of transcription Oct4?

TF for its own synthesis that matches the downstream factor

what are embryonic stem cells?

indefinite self-renewal, pluripotentcy

controlled by for TF network

these re-expressing factros reprogram adult cells inducing pluripotent stem cells

where does RNA splicing start?

5’ GU start

3’AG end

what is the sliceosome composed of?

small nuclear RNA’s (snRNA and associated proteins snRNP’s)

what do the snRNPs recognize?

they recognize splice sites at intron exon boundaries (GU-AG rule)

this assembles a step-wise process on pre-mRNA bringing exons together

catalyzes two transferication rrxn intron releases and exons joined

dynamic complex components are recycled for multiple splicing events

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translation

what are ribosomal proteins?

synthesizes in the cytoplasm and imported into the nucleus

RNA genes are clustered i the nucleolus and transcribed by RNA pol.I

what does the nucleolus act as?

acts as the cells ribosome factory

what is the structure of the euk. ribosome?

4 distinct rRNA’s

2 subunits

small-18s

large- 28s, 5.8s, 5s

where are the five “cut” locations of RNA processing?

1 and or 5

2 or 5?

what are the nucleotide modifications?

methylation

conversion or uridine→pseudouridine

preformers by snoRNPs

what are the properties of the genetic code?

redundant

conservative

unambiguous

universal

what is the start codon?

AUG

(methyionine)

what are the stop codons?

UAA, UAG, UGA

what is the role of tRNA’s?

decoding the codon

they match mRNA codon w/ amino acid it codes for

each linked to a specific amino acid

recognizes the mRNA via the anticodon

what is the unique structure of tRNA’s and why is it shapped like that?

complimentary intrachain base pairing

cloverleaf

• anticodon loop:pairs w/ codon in mRNA

• AA acceptor arm 3” CCA sequence: binds the amino acid

what is the wobbble hypothesis?

how multiple codons code for single amino acid

interchangibility of base in 3rd position

how does the tRNA recognize amino acids?

depends on aminoacyl-tRNA-synthatases that are unique to every amino acid

they covalenty link amino acids to tRNA 3’ end

what is initiator tRNA?

newly made proteins all have methionine as first a.a. at N-terminal (that is later removed in post translation modification)

→the only amino acid that can bind to P site when large ribosomal subunit is not present

what is tRNA charging?

aminoacyl-tRNA-synthetases attach the correct amino acid to its tRNA

**tRNA’s translate codons into amino acids

accuracy depedns on proper charging and anticodon pairing

initator tRNA sets reading frame for translation

give an overview of translation

1. initiation: ribosome assembles on mRNA and finds the start codon

2. elongation: amino acids are added as ribosomes moves along mRNA

3. termination: stop codon reached, completed polypeptide is released

**requires GTP and protein factors

**occurs on ribosomes in the cytoplasm

how does translation in bacteria differ from euk.?

no 5’ cap

only 3 initiation factors

no scanning step (assembles directly at start codon)

what does the info stored in the mRNA determine?

the sequence of aminoacyl-tRNAs that the ribosome accepts

what are the binding sites on the ribosome?

A(aminoacyl): binds incoming aminoacyl-tRNA carrying new a.a.

P(peptidyl): holds the tRNA w/ the polypeptide

E(exit): release empty tRNA

explain the steps of the synthesis from the ribosome

1. the second aminoacyl-tRNA binds to A site

• GTP hydrolysis releases EF-TU or eEFIA positioning to new A site

2. catalyzed by peptidyl transferase

• growing peptide is transferred from P-site tRNA to a.a. on the A-site

—after bond formation—

3. translocation

• binding of an elongation factor and GTP hydrolysis

• ribosome shifts nucleotides (one codon) in the 5’ →3’ direction

4. release of deacylated tRNA

• leaves the ribosome, emptying the E-site

what are the steps of termination?

occurs at stop codon

requires release factors → recognizes stop codons

alter the ribosomal peptidyl transferases

dissasociation of the mRNA from the ribosome, diassembly of the ribosmes

what does quality control take place?

nonsense-mediated decay (NMD)

mRNA surveillance pathway that detcts transcription with premature stop codons

degrades faulty mRNA’s to prevent production of shortened nonfunctional proteins

different mutations