Biol 213 Chapter 7

In addition to its role as the genetic material that is inherited each generation, DNA also encodes instructions for

cellular function: DNA makes RNA makes Protein

One big difference between cells is the

type and amount of proteins they contain

• This can be regulated at many different steps in gene expression

The chemical structure of RNA differs slightly from that of DNA:

1) Ribose, not deoxyribose. The extra OH makes a big difference!

2) Uracil substitutes for thymine because oxidative deamination of C produces U, and it would not be recognized as a mutation.

3) RNA mostly occurs as a single nucleotide chain, although parts of it can be folded into secondary structures.

RNA molecules can fold into specific structures that are held together by

hydrogen bonds between different base pairs

mRNA

codes for proteins

rRNA

form the core of the ribosome and catalyze protein synthesis

miRNA

regulate gene expression

tRNA

serve as adaptors between mRNA and amino acids during protein synthesis

other small RNAs

used in RNA splicing, telomere maintenance, and many other processes

transcription is key to

• understanding why different cells in a plant or animal have different properties

• understanding how levels of specific proteins are regulated

• understanding how many signals and drugs affect cells

• increasing yields of a protein in biotechnology

• decreasing levels of an enzyme to for metabolic engineering

• thinking of ways to turn off a ‘bad’ gene in medicine

transcription provides ____ of genetic information

amplification

genes can be transcribed with very different

efficiencies

• levels of gene expression are tissue-specific

sense, or coding strand

By convention, DNA is drawn with the 5’ end on the left side of the top strand. This places the strand with the same sense as the RNA on top.

• The bottom strand (running from 3’ to 5’) is what RNA polymerase actually copies into RNA

• start in direction where promoter sequence is

On an individual chromosome, either DNA strand can be

used as a template(but not at the same time)

in transcription, RNA is synthesized by

RNA polymerase

• Nucleotides added to 3’ end of RNA strand (5’ to 3’)

• RNA sequence is dependent on complementary base pairing (A-U, G-C)

• no primer, helicase, or topoisomerase needed

RNA polymerase error rate is

in 10⁴ Compare to DNA pol

Genes can be simultaneously transcribed by many

RNA polymerase molecules

why is this a bacterial cell

because of the dark little dots connected to mRNA and many ribosomes present

What controls where RNA polymerase initiates and terminates transcription?

promoters and terminators

promoter

DNA sequence that is recognized by RNA polymerase as a start point.

Chain elongation occurs until RNA polymerase reaches a _____, at which RNA is released and the RNA polymerase dissociates from the DNA

terminator site

• almost always transcribed, allows termination

similarities for transcription in bacteria and eukaryotes

1. inititation

2. elongation

3. termination

what do bacteria have in transcription that eukaryotes dont

sigma factor (eukaryotes have transcription factors instead)

sigma factor

subunit of RNA polymerase Responsible for the recognition of the promoter sequence and the tight binding of the RNA polymerase to the DNA

Producing mRNA molecules is more complex in

eukaryotes

• Multiple stages of gene expression in eukaryotes. Each stage offers opportunities to regulate expression.

• Which stage of gene regulation occurs before transcription? epigenetics regulation

Bacterial Transcription Initiation

The sigma subunit of bacterial RNA polymerase recognizes the -10 and -35 sequences and directs the rest of the RNA polymerase to bind there. Sigma factor is released after transcription begins.

ex. -10 = 10 bases before we start

• negative because zero is first base at start

Eukaryotic promoters contain sequences that promote the binding of the general transcription factors. The only one we will worry about is the

TATA box at -30, relative to the transcription start site

Eukaryotic Transcription Initiation

• RNA Pol II requires general transcription factors

• TATA-binding protein (TBP) is a subunit of TFIID; involved in the recognition of the promoter

• Assembly of transcription initiation complex; enables the recruitment of RNA Pol II

• Phosphorylation of RNA Pol II by TFIIH, releases RNA Pol II from the transcription initiation complex and allows transcription proceeds

transcription is not initiated without

TFIIH (transcription factor 2)

transcription factor 2 order

D first then that recruits B then H

Transcriptional Elongation

RNA Polymerase Unwinds DNA to access the template strand

• Only exposes ~10-20 DNA nucleotides at a time.

• Connects RNA nucleotides using DNA as a template.

• Produces the RNA transcript in a 5’ to 3’ direction

• Typically producing the RNA transcript at ~40 nucleotides per second

Bacterial transcriptional terminators often have extensive secondary structure just before the

stop side

the mechanisms of termination are different in

prokaryotes and eukaryotes

prokaryotic termination

• Rho- dependent or independent termination.

• Rho – factor or G-C hairpin

• RNA polymerase reads through a “termination sequence”

• This cause the RNA polymerase to dissociate from the DNA.

• The RNA is immediately ready for translation….boom

Eukaryotic termination

• RNA polymerase reads through a special termination sequence known as a Polyadenylation sequence (AAUAAA)

• The end of RNA transcript is then bound by proteins causing the RNA polymerase to dissociate from the DNA

• after termination, the RNA needs additional processing

RNA polymerase during transcription for prokaryotes

have a single type but differing sigma factors for specificity

RNA polymerase during transcription for eukaryotes

have three types

• RNA pol I: most rRNA genes

• RNA pol II: protein-encoding genes (makes mRNA)

• RNA pol III: tRNA, 5S rRNA, small structural RNA genes

initiation during transcription for prokaryotes

RNA pol has the sigma factor protein subunit

initiation during transcription for eukaryotes

RNA pols require general transcription factors

Transcript processing during transcription for prokaryotes

transcripts are generally NOT processed

Transcript processing during transcription for eukaryotes

mRNAs are processed

is Packing of DNA into nucleosomes in eukaryotes or prokaryotes

eukaryotes

Transcription takes place in the ___ in eukaryotic cells

nucleus

translation (protein synthesis) takes place in the _____ in eukaryotic cells

cytosol

Eukaryotic RNA must be transported from nucleus to cytosol before

leaving the nucleus, mRNA must be processed

RNA capping in eukaryotic cells

modification of 5’ end, 7-methylG

Polyadenylation in eukaryotic cells

modification of 3’ end, polyA tail

• longer tail= shorter time

splicing in eukaryotic cells

removal of introls

Prokaryotic RNA is generally not

processed

Phosphorylation of RNA Pol II by TFIIH also allows

RNA processing proteins to assemble on its tail

Capping and polyadenylation increases

stability of eukaryotic mRNA molecules

Gene organization differs between eukaryotes and prokaryotes

For most bacterial genes, the DNA sequence that encodes them is co-linear with the RNA that is produced. In contrast, most eukaryotic genes have non-coding, intervening sequences, or introns, that must be removed before a functional mRNA is made.

Introns are removed by

RNA splicing

An entire gene is transcribed (exons plus introns) but removal of the introns begins immediately after

capping occurs

• Capping and splicing both occur while RNA polymerase continues to transcribe DNA

RNA splicing is performed largely by

catalytic RNA molecules (small nuclear RNAs, snRNAs) with help from a few proteins (snRNPs). Spliceosome

• The splicing RNA molecules recognize intron- exon boundaries or junctions (sequence-specific recognition – only a small amount of bases) and cut out introns as a lariat structure

spliceosome

ribonucleoprotein complex

The sequence of only a few small parts of an intron are critical for

splicing

An intron in a pre-mRNA molecule forms a branched structure during RNA splicing, and is removed by a complex structure called a

spliceosome

Small nuclear ribonuclearproteins (SnRNPs) carry out

different stages of intron splicing

Exons are ligated back together precisely and marked with proteins (red box) to indicate

splicing has been completed

Some pre-mRNAs undergo alternative RNA splicing to produce

different mRNAs and proteins from the same gene.

• ex. striated muscle mRNA, smooth muscle mRNA, brain mRNA, etc.

rRNA is transcribed and processed in the

nucleolus

Ribosomes and mRNAs travel from the nucleus to the cytoplasm through

nuclear pores

Mature mRNAs are selectively exported from the nucleus

Only mature mRNA is exported - no introns, broken strands or incorrectly spliced variants. These are distinguished by the binding of SSspecific proteins associated with each step of RNA processing:

• polyA-binding proteins

• Cap-binding complex

• Nuclear transport receptor

In bacteria, transcription and translation are closely coupled because ribosomes can

access mRNAs are they are being transcribed

translation is key to

• understanding, and predicting the effects of, many mutations

• genetic engineering - making a precise modification to a protein

• understanding the mechanism of action of many antibiotics

template for translation

mRNA

template for transcription

DNA

There are no tRNA for stop codons, thus

61 tRNA

The trinucleotide code has

redundancy Third position “wobble”

all amino acids are encoded by multiple codons except for

Methionine and Tryptophan

how do you read this

read inside to out

stop codons

3 total

• UAA

• UAG

• UGA

Transfer RNAs (tRNAs)

adapter molecules that link codons with amino acids

hydrogen holds the

anticodon to codon bonds

The wobble position of a codon is its third base, but this corresponds to the

first base of the anticodon.

• remember the antiparallel annealing of oligonucleotides

transcription

is only the addition of nucleotides

tRNA adds anticodons from

3’ to 5’ while mRNA is in 5’ to 3’

Aminoacyl tRNA synthetases

bind to tRNAs and charge them with the appropriate amino acid.

tRNA synthetase

recognizes nucleotides at the anticodon and the 3’ amino acid-accepting arm to provide specificity. Need a different enzyme for each amino acid

The ribosome is a

ribonucleoprotein complex

eukaryotic ribosome

80S

prokaryotic ribosome

70S

Protein synthesis takes place at the

ribosome

The two ribosomal subunits come together on an ___ during protein synthesis

mRNA molecule near its 5’ end (small and large subunits)

Small ribosomal subunit

matches tRNAs with codons

large ribosomal subunit

catalyzes formation of peptide bonds

Three binding sites of protein synthesis

• A site

• P site

• E site

A site (aminoacyl-tRNA)

charged tRNA binds to its codon

P site (peptidyl-tRNA)

condensation of amino acids

E site (exit)

where the “uncharged” tRNA is ejected

RNA can be translated in three possible reading frames depending on

where the decoding process begins

• this means DNA can provide 6 possible reading frames

• the site of translation inititation is crucial!

the first peptide bond is formed in

translation elongation

Translation takes place in a

four-step cycle, which is repeated over and over during the synthesis of a protein

ribozyme

an RNA molecule that possesses catalytic activity

• Ribosomes are ribozymes, because the catalytic activity that forms peptide bonds is the rRNA molecule

Proteins are synthesized on

polyribosomes

• It is not an accident that these look like circular mRNAs, as proteins at the 5’ cap and the 3’ polyA tail interact with initiation complexes to ensure ribosomes translate only intact mRNAs.

Release factors bind stop codons triggering the release of

the newly synthesized protein

• the ribosome then dissociates from the mRNA

translation initiation study guide

• Translation begins with the codon AUG, which codes for methionine.

• An initiator tRNA molecule charged with methionine (distinct from the other methionine-carrying tRNA) binds to the P site of a small ribosomal subunit. It is the only tRNA that can bind directly to the P site, and this binding is assisted by proteins called translation initiation factors

• The loaded ribosomal subunit binds to the 5’ end of an mRNA, recognizing the cap, and moves 5’ to 3’ along the mRNA until it encounters an AUG codon (Kozak sequence).

• The initiation factors dissociate making room for the large subunit to bind forming a complete ribosome

Many proteins require post-translational modifications to become

fully functional