Transcription and Translation (Midterm 3 Lecture 2)

51 slides

what synthesizes rna transcripts, and which rna is the rna transcript in prokaryotes and eukaryotes

rna pol transcribes dna to produce rna transcript

pro = mRNA

euk = pre-mRNA (further processed to become mRNA)

what translates mRNA sequence to synthesize a polypeptide

ribosomes and tRNA (transfer rna)

what is rna polymerase, promoters, and terminators

notes:

• rna pol is diff from dna pol → rna pol transcribes, dna pol dubpicates dna

• promoters are NOT the same as a start codon → they are upstream from them

• same w terminators and stop codons → terminators are downstream

explain the initiation phase of transcription in prokaryotes

• rna pol binds to promoter sequence

  • sigma factors bind to rna pol to form the holoenzyme

• region of dna is unwound to form an open promoter complex

• unwinding lets phosphodiester bonds form btwn first 2 ribonucleotides

  • no primer needed

    • only template, can start new rna molecule from scratch

what is a holoenzyme composed of

sigma factors + rna pol

what does nascent mean

coming into or “future”

explain the elongation phase of transcription in prokaryotes

• sigma factor is released from rna pol (rna pol is now the core enzyme)

  • rna pol loses affinity for promoter and moves along template strand (in 3’ to 5’ to synthesize new rna in 5’ to 3’)

• NTPs are added to the nascent (fture) mRNA

  • complimentary base pairing w dna

  • uracil is incorporated insted of thymine (both pair w A)

• nucs are added in the 5’ to 3’ direction

why do framshifts in one gene not affect frameshifts in other genes in translation/transcription

bc whole there are non-transcribed (spacer) regions of dna → so each gene is transcribed separately

explain the termination phase of transcription in prokaryotes

transcription stops when terminator sequence is transcribed

what are the 2 kinds of terminators in prokaryotes? explain them

extrinsic: require rho factor

• rho protein binds to rna sqeunece that is C-rich and G-poor without secondary structure (on its own, no dna or anything bound to it (bc rna is single-stranded))

intrinsic: (dont require additional factors)

• no protein involved

• GC-rich rna region makes a hairpin structure when mRNA is transcribed → the stucture pulls rna away from polymerase

what are some diffs between transcription in prokaryotes vs eukaryotes

both have promoters BUT only eukaryotic have enhancers (which can be thousands of bps away from promoter (which is immediately upstream of the gene)

t/f: mRNA and protein sequences can always be directly deduced from the dna sequence

false → only in prokaryotes (“what you see is what you get” → euks have additional stages of processing after initial transcription

what are introns and which types of organisms have them

segments of dna that are transcribed but not translated

• ONLY in euk genes (not proks)

what are some modifications of primary rna transcript to produce mRNA in euks

• rna is spliced to remove introns (non-coding regions of dna)

• 5’ methyl cap is added

• poly-a-tail is added

how is the 5’ methyl cap added on eukaryotic mRNA made and what does it do

capping enzyme → adds guanidine triphosphate (the nuc) in reverse oriegntation to the 5’ end after polymerization of the transcript’s first few nucleotides

• that guanine is NOT encoded for by the gene

methyl transferases then add methyl groups to the backwards G and one or more of the succeeding nucs in the RNA

• it is critical for efficient translation, transportation out of nucleus, and preventing degredation by exonuclease (things that break down nucleic acids)

how is the poly-a-tail added on eukaryotic mRNA made and what does it do

adition of 100-200 adenosines to the 3’ end of the RNA

• NOT encoded for by the gene

• located 11-30 nucs downstream of an AAUAAA sequence

ribonuclease (RNase → cuts rna) cleaves primary transcript at terminator sequence then poly-a polymerase adds As to the 3’ end

• critical for efficient translation, transportation out of nucleus, and preventing degradation by exonuclease (similar fx to 5’ methyl cap)

what are exons

sequences found in both a gene’s dna and the mature mRNA

• exons contain coding sequences for protein product and 5’ and 3’ UTRs (untranslated regions)

what are introns

sequences dfound in a gene’s dna but NOT in the mature mRNA → they’re removed from the primary transcript through splicing

how are introns removed

through 2 sequential cuts

• one end is cut by splicosome (usually, sometimes rna transcripts are self-splicing)

• intron folds in on the other end of itself

• the other end us cut

• the mRNA fuses back tg and the spliced intron is degraded

do al euk genes include introns

no → some include multiple, some have none

what is the dystrophin gene? explain how it relates to splicing

the longest known human gene → goes from 25,000,000 bps to 14,000 after pslicing (extreme ex, usually not THIS much)

the human genome contains around 28,000 genes but human cells produce hundreds of thousands of diff proteins → how

alternative splicing

what does alternative splicing do

produces diff mature mRNA molecules from one gene

• may encode related proteins w diff partially overlapping sequences

• all exons of a gene can be transcribed, or a couple predetermined ones → hence how one gene can encode for multiple proteins w similar overlapping sequences

what is translation and what does it require to happen (what are the necessary components)

it is the process in which the genetic code carried by mRNA directs the synthesis of proteins

requires…

• mRNA, tRNA (w attached AA), and ribosomes

what is tRNA

short, single-stranded RNA molecules

• each tRNA is covalently bound to a specific AA → (when that happens its referred to as “charged” tRNA)

• base pairing btwn tRNA anticodon (complimentary to mRNA codon) and mRNA codon determine where an AA becomes incorporated into a growing peptide

explain the primary, secondary, and tertiary structure of tRNAs

primary: nuc sequence

secondary: base pairing of complimentary sequences within the tRNA (creates cloverleaf shape)

teritary: formed by 3d folding (L shape)

how many different codons are there for AAs

61 (plus 3 stop codons) → but there are fewer than 61 unique tRNA genes

what is wobble in translation

some tRNAs recognize more than one codon

• the 3’ nucleotide in the codon often doesn’t add specificity to the codon

• the nuc in the wobble position of the tRNA (the last one in the codon, 5’ end of tRNA molecule) is often modified

  • modified A,U,G, or C (w diff groups and stuff on them) → so it can bind to other things too since the last one usually doesn’t make a diff

which end of the tRNA molecule is the “wobble position”

the 5’ end of the tRNA molecule (bonds to the 3’ end of the RNA sequence)

note: wobble rules will be provided on exam

what is the I on the wobble rule chart

Inosine (a modified adenosine molecule)

what is the ribosome and what does it do

the site of protein synthesis

• composed of protein and RNA

  • has a large and small subunit

• recognizes mRNA features that signal the start of translation

  • coordinates movement of tRNAs to match codons in mRNA

  • supplies enzymatic activity to link AAs and move 5’ to 3’ along the mRNA (backwards to way nucleic acids have been read so far)

  • helps facilitate termination by dissociating from mRNA and the polypeptide

t/f: the sizes of ribosomal subunits differ bewteen pro and euks. explain why or y not (how big each subunit in both organism are)

what do the small and large subunits of ribosomes do respectively

small → binds to mRNA

large → has peptidyl transferase activity and catalyzes formation of peptide bonds

what are the 3 distinctive tRNA binding areas on the ribosome

E, P, and A sites

explain the initiation phase of translation in e. coli (prokaryotes)

Sine-Dalgarno sequence/box (on mRNA) is recognized by complementary sequences of 16 rRNA of the 30S subunit

fMet-tRNA is positioned in P site, complementary to AUG start codon on mRNA

large subunit binds

note: not all prokaryotes have same ribosome binding site recognition sequence as e. coli but many have sequences similar to it (have lots of purines (A and G))

how does the initiation phase of translation in e. coli (prokaryotes) differ in eukaryotes

in Euks…

• fMet is Met instead

• ribosome binding site is the 5’ methyl cap in euks not the shine-dalgarno box

• small ribosomal subunit is 40S not 30S

explain the elongation phase of translation in e. coli

• elongation factors (EFs) escort next tRNA into the A site of the ribosome

• peptidyl transferase (on large subunit) catalyzes formation of a peptide bond between carboxyl (C) terminus of formalmethionine (fMet) and the amino (N) terminus of the second AA

• as ribosome moves, fMet moves from P to E site

• new tRNA enters A site, fMet is released from E site

• process repeats

explain the termination phase of translation in e. coli

• nonsense (stop) codon is encountered (for which there is no tRNA)

• Release factor recognizes stop codon, moves into A site

• polypeptide is released from C-terminal tRNA

• mRNA, tRNA, and ribosomal subunits all dissociate from each other

what are polyribosomes

a complex of multiple ribosomes translating the same mRNA

• allows simultaneous synthesis of many polypeptide copies from a single mRNA

what is post-translational processing? give some exs

note: dont need to memorize exs

modifications that occur to protein after translation

• eg enzymatic cleavage (remove bits and pieces of AA)

• eg addition of chemical constituents (eg phosphorylation, glycosylaton, etc)

what are diffs in transcription/translation in pro and euks

proks:

• no nucleus so transcription and translation happen in same cellular compartments (and translation is often couples to transcription)

• genes are not divided into exons and introns

euks:

• nuc is seperated from cytoplasm by a nuclear mem

  • transcription takes place in nucleus, translation in cytoplasm → so direct couping of transcription/translation is not possible

• dna of gene consists of exons seperates by introns so the exons are defined by posttranscriptional splicing which deletes the introns

what are the diffs in prok and euk’s RNA polymerases

pro → has one RNA pol consisting of 5 subunits

euk → has 3 diff types of RNA pol, each w 10 or more subunits

• diff rna pols transcribe diff types of genes

what are the diffs in prok and euk’s RNA promoters

proks → have accessible promoters

euks → promoters can be wound up as chromatin

• enhancers clear histones from promoter and stabilize rna pol at the promoter

what are the diffs in the primary transcripts in proks and euks

proks → mRNA has triphosphate start at 5’ end and no tail at 3’ end

euks → primary transcript undergoes processing to produce mature mRNA

• adding 5’ methyl cap and poly-a-tail

• splicing out introns

how does the initiation of translation differ between proks and euks

proks:

1. unique initiator tRNA carries N-formylmethionine (fMet)

2. mRNAs have multiple ribosome bining sites (RBSs) → direct synthesis of several diff polypeptides

3. small ribosomal subunit binds immediately to mRNA’s ribosome binding site

euks:

1. initiator tRNA carried methionine (Met)

2. mRNAs have only one start site → direct synthesis of only 1 kind of polypeptide

3. small ribosomal subunit binds first to methylated cap at 5’ end of mature mRNA and then scans mRNA to find ribosome binding site

what are silent, missence, nonsense, and frameshift mutations? what do they all result in

all result in new alleles (even if silenc bc new sequence is diff)