Cell Bio- Rna: Transcription, Translation

Front 1

Structure of RNA:

Back 1

- Similar to DNA
- Polynucleotide chain
- Usually contains only one polynucleotide chain (strand)
- Pentose ribose (instead of deoxyribose)

Front 2

Bases in RNA:

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A (adenine) and G (guanine): purine bases
C (cytosine) and (U) uracil: pyrimidine bases

Front 3

Bond between nucleotides in RNA:

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- The same as in DNA
- Phosphodiester bond between 3`carbon of ribose of one nucleotide, and of 5`carbon of the second ribose nucleotide
- The polynucleotide chain has a polar character (3` end and 5` end)

Front 4

The polynucleotide chain of RNA can form more 3-D conformations. How?

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Within some parts of the RNA chain, intermolecular complementary base pairing of bases (A- U, C- G) can take place and thus double stranded segments can originate.

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mRNA:

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- Messenger RNA
- RNA transcribed from DNA that encodes relevant proteins.
- After subsequent processing it functions as a template for translation

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rRNA:

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- Ribosomal RNA
- Several types of rRNA
- 4 types in eukaryotes
- Together with proteins form ribosomes.

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mRNA in eukaryotes:

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Carries information transcribed from just one gene and codes for one protein

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mRNA i prokaryotes:

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Carries information transcribed from a set of adjacent genes and codes for several different proteins

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tRNA:

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- Transfer RNA
- During translation it functions as an adaptor recognizing corresponding codon for the amino- acid which it carries.

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Small RNA:

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Used in pre- mRNA splicing and transport of proteins to the ER

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Other roles of RNA:

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Structural and catalytic functions.

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Transcription:

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Copying genetic information from DNA, where it is saved as a sequence of bases (A, T, C, G) onto RNA, where it is also saved as a sequence of bases (A, U, C, G).
In RNA U instead of T.

Front 13

Complementary pairing:

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A new strand RNA is synthesized according to the template strand of bases of complementary pairing. Thus the sequence of bases of the RNA strand corresponds to the sequence of bases in coding DNA strand. (U in RNA instead of T in DNA)

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RNA polymerase:

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- Synthesizes the new RNA strand according to the DNA template strand in the same way as DNA polymerase does in DNA replication.
- Difference: RNA polymerase can start synthesis of new strand (do no require primer)
- Phosphodiester bond is formed between the nucleotides

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3 types of RNA polymerase in eukaryotic cells:

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- RNA polymerase I: the transcription of genes for most rRNAs. (Except 5S RNA)
- RNA polymerase II: the transcription of genes encoding proteins and genes of some small RNAs
- RNA polymerase III: the transcription of genes encoding tRNA, 5S RNA and some small RNAs.

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How does transcription start?

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By binding of RNA polymerase to the promoter of the gene which is transcribed

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Promoter:

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- A sequence of DNA which indicates the point in which transcription starts.
- The promoter is located before the first transcribed nucleotide.
- The promoter is asymmetrical and it always binds RNA polymerase on only one direction.

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Template vs. coding strand:

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DNA is read in 3`- 5` and RNA strand is synthesized in 5`- 3`. The strand which is not transcribed (RNA is 1 strand) is called the coding strand.
Because RNA polymerase can only transcribe DNA strand in 3`- 5`direction only proper strand, template strand is used.

Front 19

What regulates the binding of RNA polymerase to the promoter in eukaryotic cells?

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Specific transcription factors which is bound to the promoter.

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Transcription (continued):

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- After binding, RNA polymerase unwinds a short segment of the DNA double helix. (Making the template accessible for transcription)
- Then RNA polymerase ads the first transcribed nucleotide complementary RNA nucleotide and thus transcription is started.
- RNA polymerase continues unwinding and synthesizing
- Thus, during transcription a small hybrid double stranded DNA- RNA segment is being made. However newly synthesized RNA is quickly released
- Reaching of a terminator gene on DNA template results in the release of RNA polymerase and newly synthesized RNA strand.

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Frequency of transcription errors?

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1 error in 10 to the 4th nucleotides

Front 22

Where does post-transcriptional RNA processing take place in eukaryotic cells?

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In the nucleus, before mRNA is transported into the cytosol where it is translated.

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2 RNA processing processes that happens in both ends of the RNA strand:
Function of this:

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1. RNA capping: 7- Methylguanosine is bound to the 5`end by the unusual 5`--> 5`linkage via a bridge made of 3 phosphates (cap)
2. RNA polyadenylation: repeated adenine nucleotides are bound to the 3` end at a total extend of 100- 200 nucleotides. (poly- A- end)
- Increases the stability of mRNA.

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RNA splicing:

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Removing of noncoding sequences (introns) and joining of coding sequences (exons) in given order before transported to the cytoplasm.

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Introns vs. exons: (amount in one gene)

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Introns: 5000- 20 000 nucleotides
Exons: 1000 nucleotides

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Splicing is carried out by what?

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Large complexes of ribonucleoproteins and proteins known as spliceosomes.

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Spliceosome consists of?

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- Ribonucleoproteins contains small RNAs known as small nuclear RNAs (snRNA). These small RNAs recognize exon- intron boundaries and they form "small nuclear ribonucleoprotein particles (snRNPs) (snurps)
Snurps form the core of spliceosomes

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Mechanism of RNA splicing:

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- Spliceosome interacts with RNA at the place of relevant intron, and form a loop.
- The free ends of the neighboring exons are joined
- Loop of intron are released with the components of the spliceosome

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Evolutionary significance of introns existence:

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-Possibility of alternative splicing (more proteins from one gene)
- Increased probability of genetic recombination between exons of different genes.

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Translation:

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The synthesis of new polypeptide chain (protein) according to genetic information saved in corresponding mRNA.

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What determines the sequence of amino- acids in the polypeptide chain?

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By the sequence of bases in the corresponding mRNA

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Where and how is translation performed?

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- Translation is performed on the ribosomes.
- Translation apparatus read mRNA in 5`- 3`direction.
- New polypeptide chain is synthesized from N- end (free amino- acid with NH2 goup) towards the C- end (free amino- acid

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Composition of eukaryotic ribosome: (80S)

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- Small subunit: one type of rRNA (18S) & 33 proteins
- Large subunit: three types of rRNA (5S, 5,8S, 28S) & 49 proteins

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Specific binding sites on ribosomes:

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- There are one mRNA binding site and three tRNA binding site:
A (aminoacyl tRNA) site: it binds tRNA carrying relevant amino- acid.
P (peptidyl tRNA) site: it binds peptidyl tRNA (peptide bound to RNA)
E (exit) site: tRNA is released from the ribosome here

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tRNA structure: (5)

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1. Lenght of 80 nucleotides
2. Contains some minor bases and nucleotides derived from them (pseudouridine and dihydrouridine
3. 4 short double stranded segments formed on bases formed on complementary bases
4. Clover leaf character where three of the double stranded segments terminates with a single stranded loop.
5.Relevant amino- acid is bound at the 3`end where the terminal sequence CCA is localized by energy rich bond. Energy from this bond is used in the formation of peptide bonds during translation

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Anticodon:

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A sequence of 3 nucleotides localized on one of the arms of the tRNA molecule. They are complementary to the triplet (codon) on mRNA encoding amino- acid carried by the tRNA

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Why are only 31 types of tRNA sufficient for 20 amino-acids encoded by 61 triplets of bases?

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Because some tRNAs only require two precise complementary bindings of tRNA to codon of mRNA during translation.

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Which enzyme is responsible for binding of amino-acid to relevant tRNA with corresponding anticodon?

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aminoacyl- tRNA synthase. Each amino- acid has its own synthase enzyme. Thus synthases carry out decoding of the genetic code.

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Mechanism of translation:

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1. Translation of mRNA always starts with the sequence AUG which encodes methionine (all polypeptides starts with methionin but methionin is often removed later)
2. Translation is initiated by a tRNA carrying methionine which binds to initiator factors to the small ribosomal subunits.
3. Mentioned complex binds to 5`end of mRNA (recognizes the cap) and slides along the mRNA in 5`- 3`direction until it finds first AUG codon.
4. The large subunit binds and the ribosome is completed while the initiator tRNA binds at the P- site.
5. Aminoacyl tRNA with the anticodon corresponding to the second mRNA codon after AUG binds to the A- site--> begins elongation of peptide chain
6. Polypeptide chain is released from tRNA at the P- site and bound with a peptide bond to the amino- acid carried by the tRNA at the A- site. (catalyzed by peptidyl transferase, which is a part of the large ribosomal subunit)
7. At the same time tRNA carrying the prolonged polypeptide chain is shifted from the A- site to the P- site, and now free tRNA is shifted from the P- site to the E- site. (A- site is freed and ready for binding of another aminoacyl- tRNA
8. tRNA is released from the E- site (and the whole cycle can be repeated)

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Termination of translation:

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When one of the three "stop codons" (UAA, UAG, UGA) reaches the A- site. These codons are not recognized by any tRNA. Protein release factors are bound instead of tRNA---> peptidyl transferase catalyzes the binding of a water molecule instead of an amino-acid, thus the polypeptide chain is terminated and released from the ribosome.

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Polyribosomes:

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The initiation of translation happens repeatedly within certain short intervals on individual mRNA molecules. Thus, several ribosomes perform translation one mRNA molecule at the same time.